Novel cyanobacteriochromes active in the far-red to near-infrared

ABSTRACT

The present invention provides a protein fusion construct comprising a far-red cyanobacteriochrome (CBCR) domain linked to a heterologous domain, wherein the far-red CBCR domain comprises a CBCR polypeptide and a tetrapyrrole chromophore. The invention also provides nucleic acids, expression cassettes, vectors, and host cells for expression of the far-red CBCR protein fusion constructs. Methods for detecting cellular components, methods for imaging biological structures, and method for modulating cellular processes using the protein fusion constructs are also provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is continuation of International Application No. PCT/US2017/018976 filed Feb. 22, 2017, which claims priority to U.S. Provisional Application No. 62/298,946, filed on Feb. 23, 2016, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant No. DE-FG02-09ER16117, awarded by the U.S. Department of Energy. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Almost all organisms at the surface of the earth use photosensory proteins to sense the ambient light environment and to tune their metabolism and behavior. Animal photoreceptors provide a basis for diverse biological responses including the entrainment of circadian rhythms and visual navigation.[1, 2] Photosynthetic organisms also utilize diverse photosensors.[3] For example, flavin-based phototropins control plant phototropism, a photobiological response first noted in antiquity and studied by Charles Darwin.[4-6] Plants also contain phytochromes, linear tetrapyrrole (bilin)-containing sensors which measure red and far-red light to control every aspect of plant biology, from seed germination and light-dependent growth and development (photomorphogenesis) to shade avoidance and flowering.[7-10] Photosynthetic and nonphotosynthetic bacteria also contain photoreceptors.[11-13] Indeed, the first photobiological response discovered in cyanobacteria, complementary chromatic acclimation (CCA), was reported within 25 years of Darwin's studies on phototropism[14] and is now known to leverage bilin-based photoreceptors to optimize light harvesting under green or red light.[15-17]

More recently, photoproteins have become critical research tools. Cell biology has been profoundly altered by the discovery and development of green fluorescent protein,[18] and light-dependent channelrhodopsins have proven equally transformational in the development of optogenetic approaches to neurobiology.[19] Phytochromes have also attracted attention as fluorescent probes,[20-26] as reagents for controlling protein-protein interactions with light,[27] in systems for light-controlled gene expression and subcellular localization,[28, 28A] and as tools for regulation of second messenger metabolism with light.[29, 30] Phytochromes are particularly appealing for application in multicellular animals due to their peak absorption in the red to far-red, partially overlapping the far-red/near-infrared (near-IR) window of optimum transparency in animal tissues.[31] Moreover, metazoans lack phytochromes, so there is no endogenous phytochrome photobiology in animals. However, counterbalancing points limit such applications of phytochromes. The minimal size for photochemically and biologically functional phytochromes is relatively large (300-500 amino acids), phytochromes are often dimeric, and many phytochromes utilize reduced linear tetrapyrrole (bilin) chromophores not present in animal cells.[32-34] Moreover, most phytochromes exhibit an unusual knotted architecture [35] that can constrain their application in fusion constructs.

Cyanobacteriochromes (CBCRs) present a possible alternative. Like the distantly related phytochromes, CBCRs use 15,16-photoisomerization of bilin chromophores (FIG. 1) to reversibly photoconvert between two states with distinct spectral and biochemical properties.[34, 36] In both CBCRs and phytochromes, the bilin is covalently attached to a conserved Cys residue via a thioether linkage (FIG. 1). However, the minimal CBCR domain is much smaller than that of phytochromes (<200 amino acids), and the three CBCRs examined to date are monomeric in solution.[37-39] Several examples have shown that chromophore assembly, peak absorption, and photoconversion are properties of isolated CBCR domains rather than of the diverse cyanobacterial signaling proteins in which such domains are found.[40-45] CBCR domains often occur in the middle of these signaling proteins and hence allow more flexible design of fusion constructs.[34, 36, 46, 47] Moreover, like phytochromes, some CBCRs can utilize biliverdin IXα (BV), a bilin chromophore present in animal cells.[48, 49] CBCRs described to date exhibit a broad range of photocycles, providing responses ranging from the near ultraviolet to the red region of the visible spectrum.[36, 47, 50] Several CBCR subfamilies are recognized,[36, 43, 45, 47, 51-54] including the green/red RcaE and CcaS CBCRs regulating CCA.[17] However, the most significant disadvantage limiting their application in large animals is the absence to date of CBCRs detecting far-red and near infrared light.[55]

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention provides a protein fusion construct comprising a far-red cyanobacteriochrome (CBCR) domain linked to a heterologous domain, wherein the far-red CBCR domain comprises a CBCR polypeptide and a tetrapyrrole chromophore. In addition, the invention provides nucleic acids, expression cassettes, vectors, and host cells for expression of the far-red CBCR protein fusion constructs.

In another aspect, the invention provides a method for detecting a cellular component. The method includes: providing a protein fusion construct in a sample, the fusion construct comprising a far-red CBCR domain and a heterologous domain specifically detecting a cellular component; exposing the protein fusion construct to far-red light or near-IR light, wherein the exposing causes fluorescence of the far-red CBCR domain; and detecting the fluorescence of the far-red CBCR domain, thereby detecting the cellular component.

In a related aspect, the invention provides a method for imaging a biological structure in a subject. The method includes: providing a protein fusion construct in or near the biological structure, the fusion construct comprising a far-red CBCR domain and a heterologous domain; exposing the protein fusion construct to far-red light or near-IR light, wherein the exposing causes absorbance by, or release of an acoustic signal or fluorescence from, the far-red CBCR domain; detecting the absorbance, acoustic signal, or fluorescence of the far-red CBCR domain; and constructing an image of the biological structure; thereby imaging the biological structure.

In another aspect, the invention provides a method for modulating a cellular process. The method includes: expressing a protein fusion construct in a cell, the fusion construct comprising a far-red CBCR domain and a heterologous signaling domain; exposing the protein fusion construct to far-red light or near-IR light; wherein the exposing increases or decreases the activity of the heterologous signaling domain, thereby modulating the cellular process.

Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Structures of bilin adducts. The indicated bilins are shown as covalent adducts, with rings designated. The numbering system is shown for the covalent adduct formed by incorporation of PCB. For other adducts, positions differing from the PCB adduct are indicated. PCB, PΦB and PVB are shown in the 15Z configuration. BV is shown in the 15E configuration. PEB lacks the 15,16-double bond. P, propionate.

FIG. 2. Phylogenetic analysis of far-red CBCRs. Maximum-likelihood phylogenetic trees are shown for bilin-binding CBCR domains (left) and their associated histidine kinase bidomains (right). Far-red CBCRs, RcaE CBCRs regulating type III CCA,[16, 17] and CcaS CBCRs regulating type II CCA[42, 44] are indicated, with experimentally characterized examples matched to their kinases with solid lines and CBCRs characterized in this work in bold solid lines. Both RcaE and CcaS exhibit protochromic green/red photocycles.[17] Key regions of the underlying sequence alignments for CBCRs and histidine kinases are presented in FIG. 3 & FIG. 4.

FIG. 3. (SEQ ID NO: 55-130) Sequence alignment of CBCR domains. Trp (W) residues conserved in far-red CBCRs are in bold. The far-red/orange CBCR cluster is in bold, and the far-red/red cluster is underlined.

FIG. 4. (SEQ ID NO: 131-204) Sequence alignment of His kinase bidomains. The presumptive phosphoacceptor His residue is in bold.

FIG. 5. Characterization of purified proteins. The indicated proteins were subjected to SDS-PAGE followed by semi-dry transfer to PVDF. Transferred proteins were visualized by Amido Black (top) and by zinc blotting (bottom; PEB is not detected).

FIG. 6A. Characterization of far-red CBCRs. Absorption spectra are shown for Anacy_4718g3 in the far-red-absorbing 15Z dark state (solid black trace) and orange-absorbing 15E photoproduct (dashed grey trace). Anacy_4718g3 was purified as an intein-CBD fusion protein. Photoconversion was reversibly triggered using far-red LEDs (728 nm) or orange light (600±20 nm).

FIG. 6B. Characterization of far-red CBCRs. Absorption spectra are shown for Anacy_2551g3 using the color scheme and light sources of panel A.

FIG. 6C. Characterization of far-red CBCRs. Absorption spectra are shown for Cyan7822_4053g2 using the color scheme and light sources of panel A.

FIG. 6D. Characterization of far-red CBCRs. Absorption spectra are shown for Sta7437_1656 using the color scheme and light sources of panel A.

FIG. 6E. Characterization of far-red CBCRs. Absorption spectra are shown for WP_016871037 as initially purified (solid black trace) and after illumination with far-red light (dashed grey trace). Estimated bilin content in the two photostates was used to calculate a spectrum for the 15Z photostate (dotted black) as described in the Results.

FIG. 7A. Additional characterization of far-red/orange CBCRs. Absorption spectra are shown for His-tagged Anacy_4718g3 in the far-red-absorbing state (solid black trace) and orange-absorbing state (dashed grey trace).

FIG. 7B. Additional characterization of far-red/orange CBCRs. Normalized photochemical difference spectra are shown for intein-CBD (solid black trace) and His-tagged (circles) constructs of Anacy_4718g3.

FIG. 7C. Additional characterization of far-red/orange CBCRs. Absorption spectra are shown for Anacy_4718g3 during incubation in darkness.

FIG. 7D. Additional characterization of far-red/orange CBCRs. Normalized photochemical difference spectra are shown for Anacy4718g3 (solid black trace), Anacy2551g3 (dashed grey trace), and Cyan7822_4053g2 (circles).

FIG. 7E. Additional characterization of far-red/orange CBCRs. Normalized absorption spectra are shown for Anacy_2551g3 (solid black trace) and Sta7437_1656 (grey trace with circles) in the far-red-absorbing state.

FIG. 7F. Additional characterization of far-red/orange CBCRs. Normalized absorption spectra are shown for Sta7437_1656 (dashed grey trace) and RcaE (solid black trace) in the red-absorbing state.

FIG. 7G. Additional characterization of far-red/orange CBCRs. Forward (solid black) and reverse (dashed grey) photochemical difference spectra are shown for WP_016871037. The reverse difference spectrum was acquired after extensive illumination, whereas the forward reaction readily proceeded to completion.

FIG. 7H. Additional characterization of far-red/orange CBCRs. Normalized photochemical difference spectra are shown for Sta7437_1656 (grey trace with circles) and WP_016871037 (black trace with squares, forward reaction).

FIG. 8A. Characterization of additional CBCRs. Absorption spectra are shown for Oscil6304_4080 before (solid black) and after (dashed grey) illumination with green light (550±35 nm).

FIG. 8B. Characterization of additional CBCRs. Normalized absorption spectra are shown for the green-absorbing states of Oscil6304_4080 (solid black), green/red CBCR RcaE (dashed grey), and green/blue CBCR Oscil6304_4336g2 (solid grey with circles).

FIG. 8C. Characterization of additional CBCRs. Normalized photochemical difference spectra are shown for Oscil6304_4080 (solid black) and RcaE (dashed grey).

FIG. 8D. Characterization of additional CBCRs. Absorption spectra are shown for Nos7524_4790 in the 15Z (solid black) and 15E (dashed grey) configurations. Photoconversion was triggered using violet and red light (400±35 nm and 650±20 nm, respectively).

FIG. 8E. Characterization of additional CBCRs. Normalized absorption spectra are shown for the red-absorbing states of Nos7524_4790 (solid black) and RcaE (dashed grey).

FIG. 8F. Characterization of additional CBCRs. Absorption spectra are shown for Nos7524_4790 in the red-absorbing state after dilution into acidic guanidinium chloride. Spectra are shown before (dashed grey) and after (solid black) illumination with white light.

FIG. 9A. Assignment of biliprotein chromophore structure using acidic denaturation. Assignment of biliprotein chromophore structure using acidic denaturation. A cartoon schematic shows effects of denaturation on protein structure and reversibility of photoconversion. Denatured 15E bilin can be photoconverted to the 15Z configuration by white light in the absence of protein structure.

FIG. 9B. Assignment of biliprotein chromophore structure using acidic denaturation. Examples of denatured spectra are shown for the indicated bilins under denaturing conditions (corresponding to the lower left-hand corner in panel A). Peak wavelengths for the two chromophore bands are shown for each, along with the ratio of intensities for the two bands. Where the 15,16-double bond is present, bilin spectra are for the 15Z configuration. Covalent adducts are in solid black, and noncovalent species (PCB only) are in dashed grey.

FIG. 10A. Assignment of chemical configuration in far-red CBCRs. Absorption spectra are shown for Anacy_4718g3 in the far-red-absorbing state after dilution into acidic guanidinium chloride. Spectra are shown before (circles) and after (solid black trace) illumination with white light. Asterisk, porphyrin contaminant.

FIG. 10B. Assignment of chemical configuration in far-red CBCRs. Absorption spectra are shown for Anacy_4718g3 in the orange-absorbing state using the same assay. Spectra are shown before (dashed gray trace) and after (solid black trace) illumination with white light. Asterisk, porphyrin contaminant.

FIG. 10C. Assignment of chemical configuration in far-red CBCRs. Absorption spectra are shown for Sta7437_1656 in the far-red-absorbing state using the same assay. Spectra are shown before (circles) and after (solid black trace) illumination with white light. Asterisk, porphyrin contaminant.

FIG. 10D. Assignment of chemical configuration in far-red CBCRs. Absorption spectra are shown for Sta7437_1656 in the red-absorbing state using the same assay. Spectra are shown before (dashed gray trace) and after (solid black trace) illumination with white light. Asterisk, porphyrin contaminant.

FIG. 11A. Characterization of far-red CBCRs under denaturing conditions. Absorption spectra are shown for Anacy_2551g3 in the far-red-absorbing photostate after acid denaturation before (circles) and after (solid black trace) 1 min illumination with white light.

FIG. 11B. Characterization of far-red CBCRs under denaturing conditions. Absorption spectra are shown for Cyan7822_4053g2 in the far-red-absorbing photostate after acid denaturation before (circles) and after (solid black trace) 1 min illumination with white light.

FIG. 11C. Characterization of far-red CBCRs under denaturing conditions. A detail view is shown for WP_016871037 as purified after acid denaturation before (dashed grey trace) and after (solid black trace) 1 min illumination with white light.

FIG. 11D. Characterization of far-red CBCRs under denaturing conditions. A detail view is shown for WP_016871037 after far-red illumination and subsequent acid denaturation before (dashed grey trace) and after (solid black trace) 1 min illumination with white light.

FIG. 12A. The far-red-absorbing state contains a covalent PCB adduct. The photochemical difference spectrum for denatured Anacy_4718g3 from FIG. 4B (black line) is compared to that of NpR6012g4 (circles). [107]

FIG. 12B. The far-red-absorbing state contains a covalent PCB adduct. The 15Z peak wavelength and intensity ratio of the long-wavelength and short-wavelength (Soret) chromophore absorption bands for denatured far-red CBCRs in the 15Z configuration (black diamonds) are compared to those of CBCRs and phytochromes in the same configuration containing the indicated chromophores (circles).[53]

FIG. 13A. Characterization of Anacy_4718g3 incorporating different bilins. Absorption spectra are shown for Anacy_4718g3 incorporating PCB (solid black trace, 740 nm peak), BV (dashed black trace, no significant chromophore bound), PEB (solid grey trace, 610 nm peak), or PΦB (dashed grey trace, 752 nm peak). PCB and PΦB are in the 15Z configuration. Spectra were normalized to the protein absorption band at 280 nm to assess relative chromophore incorporation.

FIG. 13B. Characterization of Anacy_4718g3 incorporating different bilins. Absorption spectra are shown for Anacy_4718g3-PΦB in the far-red-absorbing 15Z dark state (solid black trace) and orange-absorbing 15E photoproduct (dashed grey trace).

FIG. 13C. Characterization of Anacy_4718g3 incorporating different bilins. Photochemical difference spectra are shown for native Anacy_4718g3 incorporating PCB (solid black trace), PΦB (dashed grey trace), or PEB (circles, no photoconversion) in the color scheme of panel A. Difference spectra were normalized for chromophore content.

FIG. 13D. Characterization of Anacy_4718g3 incorporating different bilins. Normalized photochemical difference spectra are shown for denatured Anacy4718g3-PΦB (dashed grey trace) and Anacy4718g3-PCB (solid black trace).

FIG. 13E. Characterization of Anacy_4718g3 incorporating different bilins. The absorption spectrum is shown for denatured Anacy_4718g3-PEB as the solid grey trace (556 nm peak). The photochemical difference spectrum resulting from 1 min illumination with white light is shown as the black trace with circles.

FIG. 14A. Characterization of far-red CBCRs using fluorescence spectroscopy. Absorption (solid black trace) and emission spectra are shown for 15Z Anacy_4718g3. The emission spectrum with 400 nm excitation (dashed dark grey) exhibits a sharp peak at 626 nm with decay at longer wavelengths, consistent with fluorescence from contaminating porphyrin. The emission spectrum with 670 nm excitation (solid grey trace with circles) exhibits similar decay, without additional peaks.

FIG. 14B. Characterization of far-red CBCRs using fluorescence spectroscopy. Absorption (solid black trace), excitation (dashed grey trace, 755 nm emission), and emission (solid grey trace with circles, 710 nm excitation) spectra are shown for Anacy_2551g3 in the 15Z far-red-absorbing state.

FIG. 14C. Characterization of far-red CBCRs using fluorescence spectroscopy. Absorption (solid black trace), excitation (dashed grey trace, 755 nm emission), and emission (solid grey trace with circles, 715 nm excitation) spectra are shown for Cyan7822_4053g2 in the 15Z far-red-absorbing state.

FIG. 14D. Characterization of far-red CBCRs using fluorescence spectroscopy. The Stokes shift for fluorescence emission is plotted versus peak wavelength for various CBCRs. Data were fit by linear regression (r²=0.96).

FIG. 15A. Correlations between CBCR spectral properties. Native 15Z peak wavelengths for PΦB adducts are plotted versus the equivalent PCB adducts for various CBCRs. Data were fit using linear regression (r²=0.998).

FIG. 15B. Correlations between CBCR spectral properties. Extinction coefficients are plotted versus peak wavelengths for various CBCRs in the 15Z photostate. Most CBCRs follow the same general trend (black squares; data fit by linear regression with r²=0.93). Far-red CBCRs (dashed box), green/red CBCRs,[44] and green/blue CBCRs[54] do not follow this trend and are omitted from the regression analysis (triangles). Far-red CBCRs are boxed.

FIG. 15C. Correlations between CBCR spectral properties. Native and denatured peak wavelengths are plotted for Anacy_4718g3 containing PCB, PΦB, and PEB in the dark-adapted state (15Z for photoactive chromophores). Data were fit by linear regression (r²>0.999).

FIG. 15D. Correlations between CBCR spectral properties. Photoconversion-induced blue shift was plotted versus 15Z peak wavelength for a range of CBCRs exhibiting trapped-twist, blue-shifted photoproducts with no second linkages in either photostate[45, 51, 53] (black circles). A linear correlation was observed (solid black trace; r²=0.92). These CBCRs were distinct from denatured samples (grey squares; linear fit, solid grey trace; r²=0.93) and CBCRs lacking key residues for trapped-twist photoproduct tuning (diamonds).[53, 71] Far-red CBCRs are boxed.

FIG. 16A. Characterization of Anacy_2551g3 using fluorescence spectroscopy. Excitation scans were performed at the indicated emission wavelengths.

FIG. 16B. Characterization of Anacy_2551g3 using fluorescence spectroscopy. Excitation scans at 755 nm (grey trace with circles) and 820 nm (black trace with diamonds) from FIG. 16A are shown in detail, with the 820 nm trace magnified 10-fold.

FIG. 16C. Characterization of Anacy_2551g3 using fluorescence spectroscopy. A plot of integrated fluorescence emission versus peak absorbance is shown for dilution series of Alexa750 dye (grey trace with circles) and Anacy_2551g3 (black trace with squares). The relative slopes and known quantum yield of Alexa 750 imply a fluorescence quantum yield of 0.012 for Anacy_2551g3.

FIG. 17. Domain architectures of cyanobacterial far-red-responsive histidine kinases. Jellybean domain-architecture cartoons are shown for the three known classes of cyanobacterial far-red histidine kinases: knotted phytochromes, knotless phytochromes, and two classes of far-red CBCRs.[93,108-110] For variable or repeated domains in far-red CBCRs, the range present for a given type of architecture is indicated by subscript.

FIG. 18 A. Imaging Anacy_2551g3 in phantom mouse. Image was acquired at 7 mm depth with 710 nm excitation and 760 nm emission. A Perkin-Elmer IVIS system was used to acquire the image with purified Anacy_2551g3.

FIG. 18 B. Imaging Anacy_2551g3 in phantom mouse. Image was acquired at 18.1 mm depth with 710 nm excitation and 760 nm emission. A Perkin-Elmer IVIS system was used to acquire the image with purified Anacy_2551g3.

FIG. 18 C. Imaging Anacy_2551g3 in phantom mouse. Image was acquired at 7 mm depth with 745 nm excitation and 800 nm emission. A Perkin-Elmer IVIS system was used to acquire the image with purified Anacy_2551g3.

FIG. 19A. Characterization of purified Anacy_2551g3 phantom with PACT. PA signals of CBCR in clear medium with 728 nm illumination and 588 nm illumination during several switching cycles.

FIG. 19B. Characterization of purified Anacy_2551g3 phantom with PACT. PACT images of silicone tubes filled with purified protein solution in clear media (first row) and under 8 mm of chicken tissue (second row).

FIG. 19C. Characterization of purified Anacy_2551g3 phantom with PACT. The graph shows a comparison of the signal ratios quantified from the ON state images and the differential images (DIFF). Error bars represent standard deviations (n=4). The protein signal amplitude was normalized to 10 μM. The hemoglobin concentration was 2.3 mM.

FIG. 20. Annexin-Anacy_2551g3 fluorescence imaging in HEK 293 cells.

DETAILED DESCRIPTION OF THE INVENTION I. General

The opacity of mammalian tissue to visible light and the strong attenuation of infrared light by water have contributed to growing interest in the development of far-red and near-infrared absorbing tools for visualizing and actuating responses within live cells. Disclosed herein is the discovery of cyanobacteriochromes (CBCRs) responsive to light in this far-red window. CBCRs are linear tetrapyrrole (bilin)-based light sensors from cyanobacteria distantly related to plant phytochrome sensors. These studies reveal far-red (λ_(max) 725-755 nm)/orange (λ_(max) 590-600 nm) and far-red/red (λ_(max) 590-600 nm) photoswitches that are small (<200 amino acids) and can be genetically reconstituted in other living cells. Phylogenetic analysis and characterization of additional CBCRs establish that far-red/orange CBCRs evolved after a complex transition from green/red CBCRs known for regulating complementary chromatic acclimation (CCA). Incorporation of different bilin chromophores demonstrates that the tuning mechanisms responsible for red-shifted chromophore absorption act on the A-, B-, and/or C-ring system, whereas photoisomerization occurs at the D-ring. Two such proteins exhibited detectable fluorescence extending well into the near infrared. This work extends the spectral window of CBCRs to the edge of the infrared, raising the possibility of using CBCRs in synthetic biology applications in the far-red region of the spectrum.

Photoreceptors provide cells with the ability to sense the light environment. Such proteins also provide valuable tools for imaging of living tissues in research or clinical settings, because non-ionizing visible and near-infrared light is much safer than UV or x-ray irradiation. Human or animal tissues are maximally transparent to far-red and near-infrared light. Therefore, photoreceptors able to detect this spectral window are optimal for such applications. Very few photoreceptors detect light in this region of the spectrum. As such, a new group of photoreceptors with peak absorption in the far-red and with detectable fluorescence well into the near infrared has now been identified. These new photoreceptors can be developed for a range of applications, including fluorescent reporters, optical contrast agents, and optical tomography.

II. Definitions

As used herein, the terms “nucleic acid,” “nucleotide,” and “polynucleotide” refer to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers. The term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, and DNA-RNA hybrids, as well as other polymers comprising purine and/or pyrimidine bases or other natural, chemically modified, biochemically modified, non-natural, synthetic, or derivatized nucleotide bases. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), orthologs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The terms “nucleotide sequence encoding a peptide” and “gene” refer to the segment of DNA involved in producing a peptide chain. In addition, a gene will generally include regions preceding and following the coding region (leader and trailer) involved in the transcription/translation of the gene product and the regulation of the transcription/translation. A gene can also include intervening sequences (introns) between individual coding segments (exons). Leaders, trailers, and introns can include regulatory elements that are necessary during the transcription and the translation of a gene (e.g., promoters, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions, etc.). A “gene product” can refer to either the mRNA or protein expressed from a particular gene.

“Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence (e.g., a peptide of the invention) in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

“Identical” and “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same. Sequences are “substantially identical” to each other if they have a specified percentage of nucleotides or amino acid residues that are the same (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms with manual adjustment informed by structural parameters. These definitions also refer to the complement of a nucleic acid test sequence.

“Similarity” and “percent similarity,” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of amino acid residues that are either the same or similar as defined by conservative amino acid substitutions (e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% similar over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms with manual adjustment informed by structural parameters. Sequences are “substantially similar” to each other if, for example, they are at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% similar to each other.

Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, substitutions may be made wherein an aliphatic amino acid (e.g., G, A, I, L, or V) is substituted with another member of the group. Similarly, an aliphatic polar-uncharged group such as C, S, T, M, N, or Q, may be substituted with another member of the group; and basic residues, e.g., K, R, or H, may be substituted for one another. In some embodiments, an amino acid with an acidic side chain, e.g., E or D, may be substituted with its uncharged counterpart, e.g., Q or N, respectively; or vice versa. Each of the following eight groups contains other exemplary amino acids that are conservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins, 1993).

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins, the BLAST and BLAST 2.0 algorithms and the default parameters discussed below are used.

Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or with manual adjustment informed by structural parameters (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

Additional examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1990) J Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available at the National Center for Biotechnology Information website, ncbi.nlm.nih.gov. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=−2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA, 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or peptides are substantially identical is that the peptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the peptide encoded by the second nucleic acid. Thus, a peptide is typically substantially identical to a second peptide, for example, where the two peptides differ only by conservative amino acid substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

The terms “transfection” and “transfected” refer to introduction of a nucleic acid into a cell by non-viral or viral-based methods. The nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. See, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88.

The terms “expression” and “expressed” in the context of a gene refer to the transcriptional and/or translational product of the gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell.

Expression of a transfected gene can occur transiently or stably in a cell. During “transient expression” the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell.

As used herein, the term “amino acid” refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. Amino acids include naturally-occurring α-amino acids and their stereoisomers, as well as unnatural (non-naturally occurring) amino acids and their stereoisomers. “Stereoisomers” of a given amino acid refer to isomers having the same molecular formula and intramolecular bonds but different three-dimensional arrangements of bonds and atoms (e.g., an L-amino acid and the corresponding D-amino acid).

Naturally-occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate and O-phosphoserine. Naturally-occurring α-amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of a naturally-occurring α-amino acids include, without limitation, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

Unnatural (non-naturally occurring) amino acids include, without limitation, amino acid analogs, amino acid mimetics, synthetic amino acids, N-substituted glycines, and N-methyl amino acids in either the L- or D-configuration that function in a manner similar to the naturally-occurring amino acids. For example, “amino acid analogs” can be unnatural amino acids that have the same basic chemical structure as naturally-occurring amino acids (i.e., a carbon that is bonded to a hydrogen, a carboxyl group, an amino group) but have modified side-chain groups or modified peptide backbones, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. “Amino acid mimetics” refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally-occurring amino acid.

Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, as described herein, may also be referred to by their commonly accepted single-letter codes.

With respect to amino acid sequences, one of skill in the art will recognize that individual substitutions, additions, or deletions to a peptide, polypeptide, or protein sequence which alters, adds, or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. The chemically similar amino acid includes, without limitation, a naturally-occurring amino acid such as an L-amino acid, a stereoisomer of a naturally occurring amino acid such as a D-amino acid, and an unnatural amino acid such as an amino acid analog, amino acid mimetic, synthetic amino acid, N-substituted glycine, and N-methyl amino acid.

The terms “amino acid modification” and “amino acid alteration” refer to a substitution, a deletion, or an insertion of one or more amino acids. For example, substitutions may be made wherein an aliphatic amino acid (e.g., G, A, I, L, or V) is substituted with another member of the group. Similarly, an aliphatic polar-uncharged group such as C, S, T, M, N, or Q, may be substituted with another member of the group; and basic residues, e.g., K, R, or H, may be substituted for one another. In some embodiments, an amino acid with an acidic side chain, e.g., E or D, may be substituted with its uncharged counterpart, e.g., Q or N, respectively; or vice versa. Each of the following eight groups contains exemplary amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1993)).

As used herein, the term “expression cassette” refers to a nucleic acid construct, which when introduced into a host cell, results in transcription and/or translation of an RNA or polypeptide, respectively. Antisense constructs or sense constructs that are not or cannot be translated are expressly included by this definition. One of skill will recognize that the inserted polynucleotide sequence need not be identical, but may be only substantially similar to a sequence of the gene from which it was derived.

As used herein, the term “promoter” refers to a polynucleotide sequence capable of driving transcription of a coding sequence in a cell. Thus, promoters used in the polynucleotide constructs of the invention include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5′ and 3′ untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) gene transcription. A “constitutive promoter” is one that is capable of initiating transcription in nearly all tissue types without a need for a particular stimulus or condition, whereas a “tissue-specific promoter” initiates transcription only in one or a few particular tissue types. An “inducible promoter” is one that initiates transcription only under particular environmental conditions or developmental conditions.

As used herein, the term “vector” refers to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell. An expression vector may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression vector includes a polynucleotide to be transcribed, operably linked to a promoter. Nucleic acid or amino acid sequences are “operably linked” (or “operatively linked”) when placed into a functional relationship with one another. For instance, a promoter or enhancer is operably linked to a coding sequence if it regulates, or contributes to the modulation of, the transcription of the coding sequence. Operably linked DNA sequences are typically contiguous, and operably linked amino acid sequences are typically contiguous and in the same reading frame. However, since enhancers generally function when separated from the promoter by up to several kilobases or more and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous. Similarly, certain amino acid sequences that are non-contiguous in a primary polypeptide sequence may nonetheless be operably linked due to, for example folding of a polypeptide chain.

A polynucleotide/polypeptide sequence is “heterologous” to an organism or a second polynucleotide/polypeptide sequence if it is synthetic or originates from a different species, or, if from the same species, is modified from its original form. For example, when a promoter is said to be operably linked to a heterologous coding sequence, it means that the coding sequence is derived from one species whereas the promoter sequence is derived another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence, e.g., from a different gene in the same species, or an allele from a different ecotype or variety).

The term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. For example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed, or not expressed at all.

As used herein, the terms “cyanobacteriochrome” and “CBCR” refer to sensory photoreceptor proteins that mediate one or more processes including, but not limited to, photochromic responses, phototactic responses, development, and nitrogen metabolism in cyanobacteria. CBCRs typically include at least one GAF domain and at least one bilin chromophore as described herein. Among other characteristics, CBCRs exhibit a variety of photocycles spanning the entire visible and near-UV spectrum. At least six subfamilies of CBCRs have been identified based on photochemistry and primary structure. Examples of previously known CBCRs include Tlr0924, AnPixJg2, TePixJg, NpR6012g4, CikA, CcaS, and RcaE.

As used herein, the terms “far-red cyanobacteriochrome” and “near-IR cyanobacteriochrome” refer to CBCRs having at least one local absorbance maximum and/or at least one local emission maximum in the far-red portion or the near-IR portion of the electromagnetic spectrum. “Far-red” refers to light having an emission spectrum where there is an emission peak or emission maximum at a wavelength ranging from about 720 nm to about 760 nm. “Near infra-red” and “NIR” refer to light having an emission spectrum where there is an emission peak or emission maximum at a wavelength ranging from about 761 nm to about 1000 nm.

The “bilin” components of the adducts of the present invention are linear oligopyrroles (e.g., di-, tri-, or tetrapyrroles) capable of fluorescing, or photointerconverting between spectrophotometrically distinct forms, when associated with an apoprotein. Typically, the bilin components of the invention are isolated from vascular plants, algae, or cyanobacteria according to standard techniques or are synthesized in the same cell in which a CBCR is expressed. The bilin components can also be synthesized de novo. For a general discussion of bilins useful in the present invention, see, e.g., Falk (1989) Pp. 355-399 In: The Chemistry of Linear Oligopyrroles and Bile Pigments, Springer-Verlag, Vienna. Examples of bilins include, but are not limited to, phycocyanobilin (PCB), phytochromobilin (PΦB), phycoerythrobilin (PEB), and any of the four biliverdin IX (BV) isomers: BV IXα, IXβ, IXγ or IXδ.

As used herein, the term “GAF domain” refers to a polypeptide having a characteristic tertiary structure present in a number of cGMP phosphodiesterases, certain adenyl cyclases, and the bacterial transcription factor FhlA first described by Aravind and Pontig (Tr. Biochem. Sci. 22(12): 458-459. 1997). The structure of GAF domains is described, for example, by Hurley et al. (EMBO J., 19(20): 5288-5299. 2000) and Narikawa et al. (Proc. Nat. Acad. Sci. USA, 110(3): 918-923. 2013). Characteristic features of GAF domains of the invention include a central 5-stranded antiparallel β-sheet, one or more α-helices on the chromophore-binding side of the domain, and two or more α-helices opposite the chromophore-binding side of the domain.

As used herein, the term “acidic motif” refers to a region in the chromophore-binding side of the domain containing at least one acidic amino acid residue that contributes to chromophore binding.

As used herein, the term “signaling protein” refers to a protein that is part of a cellular signal transduction pathway. Examples of signaling pathways include, but are not limited to, MAP kinase signaling, PI3K/Akt signaling, protein kinase C signaling, and phospholipase signaling. Examples of signaling proteins include, but are not limited to, kinases, phosphatases, phosphodiesterases, proteases, phopholipases, cyclase, G-proteins, and channel proteins. A “signaling polypeptide” refers to a polypeptide exhibiting signaling activity by itself or as part of a protein fusion construct.

As used herein, the term “structural protein” refers to a protein that provides structural support to cells and other biological structures. Structural proteins often assemble to form structures such as filaments, cables, and sheets to provide biomechanical properties necessary for maintenance of cell shape and function. Non-limiting examples of structural proteins include actin, tubulin, myosin, keratin, fibroin, collagen, elastin, and proteoglycans. A “structural polypeptide” refers to a polypeptide exhibiting the structural properties and/or assembly properties of a structural protein by itself or as part of a protein fusion construct.

As used herein, the term “transport protein” refers to a polypeptide which functions to convey molecules or inorganic ions (e.g., H⁺, K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl⁻, Br⁻, etc.) into (e.g., uptake proteins) and out of (e.g., efflux proteins) a cell, as well as transportation of molecules intracellularly (e.g., translocation proteins) and other related transport activity. Examples of transport proteins include, but are not limited to, annexins, clathrin, caveolins, SNARE proteins, glucose transporter proteins, and aquaporins. A “transport polypeptide” refers to a polypeptide exhibiting the activity of a transport protein by itself or as part of a protein fusion construct.

As used herein, the terms “targeting protein” and “targeting polypeptide” refer to a protein that can selectively interact with a target feature such as a cellular receptor or another cell surface protein. Examples of targeting proteins include, but are not limited to, annexins, antibodies, antibody fragments such as synthetic F_(ab)'s, aptamers, and subcellular targeting signals, such as, e.g., mitochondrial or chloroplast targeting sequences, signal sequences, ER retention sequences, nuclear localization/export sequences, and the like.

As used herein, the term “hormone protein” refers to a protein that serves as an extracellular signal to elicit a response from a target cell or tissue. Examples of hormone proteins include, but are not limited to, insulin, luteinizing hormone, and platelet-derived growth factor. A “hormone polypeptide” refers to a polypeptide that exhibits the activity of a hormone protein by itself or as part of protein fusion construct.

As used herein, the term “regulatory protein” refers to a protein that contributes to the control of cellular processes and/or physiological activity. Examples of regulatory proteins include, but are not limited to, transcription factors, corepressors, coactivators, and the like. A “regulatory polypeptide” refers to a polypeptide that exhibits the activity of a regulatory protein by itself or as part of protein fusion construct.

III. Far-Red CBCR Constructs

The present invention is directed to fluorescent protein adducts, referred to herein as far-red CBCR labels, and their use as fluorescent markers/labels in a variety of contexts. The far-red CBCR labels comprise an apoprotein component (i.e., a far-red cyanobacteriochrome) and a tetrapyrrole component (e.g., a bilin such as phycoerythrobilin (PEB)). The far-red CBCR labels (fluorescent adducts) can be chemically conjugated or fused (i.e., recombinantly expressed as a fusion protein) to a heterologous subject moiety that is to be so labeled. For example, the labeled moiety can be a member of a biological binding pair for use in a number of techniques involving fluorescent labeling of analytes or other moieties.

A. CBCR Polypeptides

Accordingly, a first aspect of the invention provides a protein fusion construct comprising a far-red cyanobacteriochrome (CBCR) domain linked to a heterologous domain, wherein the far-red CBCR domain comprises a CBCR polypeptide and a tetrapyrrole chromophore. The CBCR polypeptide generally includes at least one GAF domain, named for the presence of domain in various cGMP phosphodiesterases (G), certain adenyl cyclases (A), and the bacterial transcription factor FhlA (F).

In some embodiments, the CBCR polypeptide comprises a GAF domain having an acidic motif comprising:

-   -   a conserved tryptophan residue; followed by     -   two acidic amino acid residues, wherein at least one of the         amino acid residues is an acidic amino acid residue; followed by     -   a conserved glutamic acid residue; followed by     -   a further amino acid residue; followed by     -   an aromatic amino acid residue.

In some embodiments, the acidic motif comprises an amino acid sequence set forth in SEQ ID NO: 1:

(1) W-X⁹-X⁶-E-X¹-X⁵

-   -   wherein:     -   W is a tryptophan residue;     -   E is a glutamic acid residue;     -   X¹ is an independently selected amino acid residue;     -   X⁵ is independently selected from the group consisting of a         phenylalanine residue, a tyrosine residue, and a tryptophan         residue;     -   X⁶ is independently selected from the group consisting of an         aspartic acid residue and an asparagine residue; and     -   X⁹ is independently selected from the group consisting of an         aspartic acid residue and a glutamate residue.

In other embodiments, the acidic motif comprises an amino acid sequence set forth in SEQ ID NO: 2:

(2) X²-X¹-D-E-X¹-X²-P

-   -   wherein:     -   each X¹ is independently selected from the group consisting of         an alanine residue, a cysteine residue, a valine residue, a         threonine residue, a leucine residue, an isoleucine residue, a         methionine residue, a phenylalanine residue, a tyrosine residue,         and a tryptophan residue;     -   X¹ is an independently selected amino acid residue;     -   D is an aspartic acid residue;     -   E is a glutamic acid residue; and     -   P is a proline residue.

When complexed with the CBCR polypeptides of the invention, tetrapyrrole chromophores (e.g., bilins) exhibit large red shifts in their absorbance and fluorescence spectra as described in more detail below. Without wishing to be bound by any particular theory, it is believed that the presence of multiple conserved Trp residues in the CBCR polypeptides could promote a charge-transfer process that generates a labile, red-shifted species.

In some embodiments, the CBCR polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 3:

(3) X⁹-R-X¹-X³-X⁴-F-X¹-X³-(X¹)₂-X⁶-G-(X¹)₃-X⁴- X²-E-E-X¹-V-(X¹)₃-X²-(X¹)₂-X²-(X¹)₄-W-X⁸-X⁶- E-X¹-X⁵-X¹-X⁷-X⁹-(X²)₂-X⁸-X²-Y-X¹-Q-G-X¹-P-R-I-V- X¹-X⁶-V-X²-X¹⁰-X¹-D-X¹-X⁵-X²-X¹-C-L-X¹-E-X⁵- (X¹)₅-X⁴-X¹-S-K-X⁴-V-A-P-I-X²;

-   -   wherein each A is an alanine residue, each C is a cysteine         residue, each D is an aspartic acid residue, each E is a         glutamic acid residue, each F is a phenylalanine residue, each G         is a glycine residue, each I is an isoleucine residue, each K is         a lysine residue, each L is a leucine residue, each P is a         proline residue, each Q is a glutamine residue, each R is an         arginine residue, each S is a serine residue, each V is a valine         residue, each W is a tryptophan residue, and each Y is a         tyrosine residue;     -   and wherein:     -   each X¹ is an independently selected amino acid residue;     -   each X² is independently selected from the group consisting of         an alanine residue, a cysteine residue, a valine residue, a         threonine residue, a leucine residue, an isoleucine residue, a         methionine residue, a phenylalanine residue, a tyrosine residue,         and a tryptophan residue;     -   each X³ is independently selected from the group consisting of a         valine residue, a leucine residue, an isoleucine residue, and a         methionine residue;     -   each X⁴ is independently selected from the group consisting of a         valine residue, an isoleucine residue, and a threonine residue;     -   each X⁵ is independently selected from the group consisting of a         phenylalanine residue, a tyrosine residue, and a tryptophan         residue;     -   each X⁶ is independently selected from the group consisting of         an aspartic acid residue and an asparagine residue;     -   each X⁷ is independently selected from the group consisting of a         glutamic acid residue and a glutamine residue;     -   each X⁸ is independently selected from the group consisting of         an aspartic acid residue, an asparagine residue, a glutamic acid         residue, and a glutamine residue;     -   each X⁹ is independently selected from the group consisting of         an aspartic acid residue and a glutamate residue; and     -   X¹⁰ is absent or X¹⁰ is one or more independently selected amino         acid residues.

In some embodiments, X¹⁰ is absent.

In some embodiments, the CBCR polypeptide comprises an amino acid sequence set forth in:

-   -   SEQ ID NO: 4 (Sta7437_1656),     -   SEQ ID NO: 5 (Cyan7822_4053g2),     -   SEQ ID NO: 6 (Anacy_2551g3), or     -   SEQ ID NO: 7 (Anacy_4718g3).

In some embodiments, the CBCR polypeptide comprises an amino acid sequence set forth in:

-   -   SEQ ID NO: 4 (Sta7437_1656),     -   SEQ ID NO: 5 (Cy7822_4053g2);     -   SEQ ID NO: 6 (Anacy_2551g3);     -   SEQ ID NO: 7 (Anacy_4718g3);     -   SEQ ID NO: 8 (N7104D_1016g3);     -   SEQ ID NO: 9 (L6406D_1154g2);     -   SEQ ID NO: 10 (c56D2_02270g2);     -   SEQ ID NO: 11 (c407D_01196g2);     -   SEQ ID NO: 12 (fdiDRAFT29700);     -   SEQ ID NO: 13 (WP009627289g3);     -   SEQ ID NO: 14 (Os7112_5903g3);     -   SEQ ID NO: 15 (C6303_3693g3);     -   SEQ ID NO: 16 (WP006632756g3);     -   SEQ ID NO: 17 (Cy7425_1390g3);     -   SEQ ID NO: 18 (WP017296986g2);     -   SEQ ID NO: 19 (WP_033374293);     -   SEQ ID NO: 20 (WP028089844g3);     -   SEQ ID NO: 21 (WP008316973g2);     -   SEQ ID NO: 22 (Ga0039499_10213);     -   SEQ ID NO: 23 (310F_3509)     -   SEQ ID NO: 24 (WP_016871037);     -   SEQ ID NO: 25 (WP_016878855);     -   SEQ ID NO: 26 (WP_026722600);     -   SEQ ID NO: 27 (WP_017309337); or     -   SEQ ID NO: 28 (WP_016873240).

In some embodiments, the CBCR polypeptide in the far-red CBCR domain has at least about 80%, e.g., at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity to any one of the amino acid sequences set forth in SEQ ID NOS: 1-28 (e.g., SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7). In some embodiments, the CBCR polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In some embodiments, the CBCR polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the CBCR polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the CBCR polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the CBCR polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7.

B. Chromophores for Far-Red CBCR Domains

As described above, the far-red CBCR domain in the protein fusion construct of the invention includes a tetrapyrrole chromophore. The far-red CBCR domain can contain any natural or synthetic tetrapyrrole capable of binding to the CBCR polypeptide in the domain. In some embodiments, the invention provides protein fusion constructs as described above wherein the tetrapyrrole chromophore is a bilin.

The far-red CBCR domain can contain one bilin, which is generally covalently coupled to the domain through one or more cysteine thioether linkages. As such, the far-red CBCR domain of the protein fusion construct provides a substrate for autocatalytic bilin addition.

Bilins and other tetrapyrrole chromophores can be isolated from natural sources or synthesized according to techniques known in the art. Methods for synthesis of the dimethyl ester of phytochromobilin are described, for example, by Weller et al. (Chem. Ber. 113:1603-1611 (1980)). Conversion of the dimethyl ester to the free acid can be accomplished according to known techniques (see, e.g., Greene and Wuts, Protective Groups in Organic Synthesis, 4th Ed. 2007, Wiley-Interscience, New York). Bilins including phytochromobilin, phycocyanobilin (PCB), and phycoerythrobilin (PEB) can be isolated from natural sources according to known methods. For instance, crude phycocyanobilin can be prepared from Spirulina platensis as described by Terry et al. (1993) J. Biol. Chem. 268:26099-26106. Crude phytochromobilin and PEB can be prepared by methanolysis of Porphyridium cruentum cells as described by Cornejo et al. (1992) J. Biol. Chem. 267: 14790-14798. The structures of phytochromobilin, PCB, and PEB are shown below. As described herein, the protein fusion constructs can also be co-expressed with enzymes that are capable of forming bilin chromophores.

Accordingly, some embodiments of the invention provide protein fusion constructs as described above, wherein the bilin is selected from the group consisting of phycocyanobilin (PCB), phytochromobilin (PΦB), phycoerythrobilin (PEB), and biliverdin (BV).

The far-red CBCR domain confers fluorescence on the fusion protein, preferably providing fluorescence quantum yield and molar extinction coefficients of at least 1%, e.g., at least 10%, 50%, 75%, 90% of a corresponding unfused far-red CBCR domain, or substantially equivalent to a corresponding unfused far-red CBCR domain, measured as described herein. Certain domains provide extinction coefficients of at least 40,000 and/or quantum yields of at least 0.01. In certain instances, the fluorescence emission spectrum of the far-red CBCR protein fusion construct is substantially equivalent to that of a corresponding unfused CBCR.

In some embodiments, the C-terminus of the CBCR polypeptide is linked to the heterologous domain. In some embodiments, the N-terminus of the CBCR polypeptide is linked to the heterologous domain.

In some embodiments, the protein fusion construct further comprises one or more ancillary amino acid sequences located at the N-terminus and/or the C-terminus of the CBCR polypeptide or the heterologous polypeptide. These ancillary sequences can be useful for expressing, purifying, and/or using the protein fusion construct. The protein fusion construct can contain, for example, a poly-histidine tag (e.g., a His6 tag); a calmodulin-binding peptide (CBP) tag; a NorpA peptide tag; a Strep tag (e.g., Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) for recognition by/binding to streptavidin or a variant thereof; a FLAG peptide (i.e., Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) for recognition by/binding to anti-FLAG antibodies (e.g., M1, M2, M5); a glutathione S-transferase (GST); a chitin-binding domain (CBP) or a maltose binding protein (MBP) polypeptide.

Various spacers or flexible linker peptides providing a variety of functionalities, such as a specific endopeptidase recognition and/or cleavage site, an affinity-purification tag, etc., can be used between the heterologous domain and the far-red CBCR domain. For example, when displayed C-terminally to the far-red CBCR domain, a specific protease recognition and cleavage site can be engineered immediately upstream from the heterologous domain so, upon cleavage with the protease, the heterologous domain can be cleanly released from the protein fusion construct. This strategy also works for most proteins displayed on the N-terminus of the fusion protein because the functions of most heterologous proteins are not affected by C-terminal extensions several residues long. In situations where such C-terminal extension is highly undesirable, an intein domain (Perler (2000), Nucleic Acids Res 28:344-345) can be engineered immediately downstream from the heterologous protein domain. Subsequent excision of intein cleanly releases the displayed domain from the fusion protein.

The length and amino acid sequence requirements of such linkers are readily determined empirically for a given fusion construct. Generally, the linkers are preferably from at least 5, preferably at least 10 residues in length, typically requiring no more than 50, and more often no more than 30 residues. To facilitate an unintrusive orientation, small, flexible residues such as Ala, Gly and Ser are particularly convenient components.

C. Heterologous Domains

A number of heterologous domains are suitable for use in the protein fusion constructs of the invention. In general, the heterologous domain will contain a polypeptide, some of which will be recognized by particular antibodies, receptors, enzymes, for use in particular applications. For example, the fusion protein construct can contain a specific binding moiety comprising at least one of a specific binding pair, such as a receptor-ligand pair, e.g., an immunoglobulin antigen-binding domain or antigenic domain, a lectin saccharide-binding domain, or an avidin or streptavidin domain. In a particular embodiment, the fusion protein comprises a biotinylated or biotinylatable domain, which is preferably biotinylated in the expression system (e.g., cell) selected for expression of the fusion protein. A wide variety of synthetic, semi-synthetic and natural such domains are known in the art, including homologs in phycobiliprotein producing cyanobacteria (see, e.g., Schatz et al. 1993, Bio/Technology 11, 1138-1143; Tatsumi et al., 1996, Anal Biochem 243, 176-180; Samols et al. 1988, J Biol Chem 263, 6461-6464; Gomicki et al. 1993, J Bacteriol 175, 5268-5272; Phung et al., GenBank Accession No. U59235; Nakamura et al. 1998 Nucl Acids Res 26, 63-67). In fact, enzymes sufficient to biotinylate biotinylatable domains have been characterized, permitting in vitro biotinylation. These biotinylated domains permit especially convenient affinity purification tags and are useful in the many well developed biotin/avidin applications (see, e.g., Beckett et al. 1999, Protein Sci 8, 921-929; Buoncristiani et al. 1988, J Biol Chem 263, 1013-1016; Li et al., 1992, J Biol Chem 267, 855-863; Cronan 1990, J Biol Chem 265, 10327-10333; Wilchek and Bayer (ed) 1990, Methods Enzymol 184).

In some embodiments, the invention provides far-red CBCR protein fusion constructs as described above, wherein the heterologous domain comprises a heterologous polypeptide selected from the group consisting of a signaling polypeptide, a structural polypeptide, a transport polypeptide, a targeting peptide, a hormone polypeptide, and a regulatory peptide.

In some embodiments, the signaling polypeptide is selected from the group consisting of a kinase polypeptide, a phosphatase polypeptide, a phosphodiesterase polypeptide, a nucleotide cyclase polypeptide, a protease, a phopholipase, a G-protein polypeptide, and a channel protein polypeptide.

In some embodiments, the structural polypeptide is selected from the group consisting of an actin polypeptide, a tubulin polypeptide, a myosin polypeptide, and a collagen polypeptide.

In some embodiments, the transport polypeptide is selected from the group consisting of an annexin polypeptide and a clathrin polypeptide.

In some embodiments, the targeting polypeptide is selected from the group consisting of an antibody, an antibody fragment, an aptamer, and a subcellular targeting signal.

In some embodiments, the heterologous domain comprises a streptavidin polypeptide.

The far-red CBCR label labels of this invention can be attached to a variety of other non-protein heterologous groups, including nucleic acids (e.g., single or double stranded DNA, cDNA, mRNA, cRNA, rRNA, tRNA) various sugars and polysaccharides, lectins, and the like. Uses of the various labeled biomolecules will be readily apparent to one of skill in the art. For example, labeled nucleic acids can be used as probes to specifically detect and/or quantify the presence of the complementary nucleic acid in, for example, fluorescence in situ hybridization or a Southern blot.

The far-red CBCR labels of this invention can be attached to non-biological molecules and various articles of manufacture. Thus, for example where it is desired to associate an article of manufacture with a particular manufacturer, distributor, or supplier, the far-red CBCR label, or simply one component of the far-red CBCR label can be attached to the subject article. Later development (e.g., by addition of the second component such as bilin or apoprotein) and exposure to an appropriate light source will provide a fluorescent signal identifying the article as one from a source of such labeled articles.

D. Recombinant Nucleic Acids and Host Cells for Expression

In a related aspect, the invention provides nucleic acids encoding the CBCR polypeptides and far-red CBCR protein fusion constructs as described herein. The nucleic acids can be generated from a nucleic acid template encoding CBCRs, using a number of recombinant DNA techniques that are known to those of skill in the art. Accordingly, certain embodiments of the invention provide an isolated nucleic acid comprising a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOS: 1-28 (e.g., SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7).

In some embodiments, the invention provides an isolated far-red CBCR nucleic acid having at least about 50%, e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity to a nucleic acid sequence encoding an amino acid sequence set forth in any one of SEQ ID NOS: 1-28 (e.g., SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7). In certain embodiments, the isolated far-red CBCR nucleic acid is a synthetic gene that uses alternative codons for encoding an amino acid sequence set forth in any one of SEQ ID NOS: 1-28.

Using a far-red CBCR nucleic acid of the invention, a variety of expression constructs and vectors can be made. Generally, expression vectors include transcriptional and translational regulatory nucleic acid regions operably linked to the nucleic acid encoding the far-red CBCR. The term “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. In addition, the vector may contain a Positive Retroregulatory Element (PRE) to enhance the half-life of the transcribed mRNA (see, Gelfand et al. U.S. Pat. No. 4,666,848). The transcriptional and translational regulatory nucleic acid regions will generally be appropriate to the host cell used to express the far-red CBCR. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. In general, the transcriptional and translational regulatory sequences may include, e.g., promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Typically, the regulatory sequences will include a promoter and/or transcriptional start and stop sequences. Vectors also typically include a polylinker region containing several restriction sites for insertion of foreign DNA. As described above, heterologous sequences (e.g., a fusion tag such as a His tag) can be used to facilitate purification and, if desired, removed after purification. The construction of suitable vectors containing DNA encoding replication sequences, regulatory sequences, phenotypic selection genes, and the far-red CBCR of interest are prepared using standard recombinant DNA procedures. In some instances, isolated plasmids, viral vectors, and DNA fragments are cleaved, tailored, and ligated together in a specific order to generate the desired vectors, as is well-known in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, N.Y., 2nd ed. 1989)). In other instances, topo cloning or cloning by gap repair in yeast can be used.

Accordingly, some embodiments of the invention provide an expression cassette comprising a far-red CBCR nucleic acid as described herein operably linked to a promoter. In some embodiments, the invention provides a vector comprising a far-red CBCR nucleic acid as described herein. In some embodiments, the far-red CBCR nucleic acid in the expression cassette or vector encodes a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOS: 1-28 (e.g., SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7).

Cassettes for expression of the CBCR polypeptides can further include one or more nucleic acids encoding enzymes that synthesize the tetrapyrrole chromophores (e.g., a heme oxygenase for forming a bilin such as biliverdin). The heme oxygenase can be native or recombinant, such as a recombinantly expressed HO1 from Synechocystis sp. PCC6803. A bilin is generally further subject to a bilin reductase and may be further subject to additional enzymes of the cell such as additional reductases, to form the particular bilin. Nucleic acids encoding a number of recombinant reductases can be included in the expression cassette and in various combinations to obtain the far-red CBCR protein fusion constructs. For example, the expression cassettes can include nucleic acids encoding 3Z-phycocyanobilin:ferredoxin oxidoreductase (PcyA), which converts biliverdin to PCB; 3Z-phycoerythrobilin:ferredoxin oxidoreductase (PebS), which converts biliverdin to 3Z-phycoerythrobilin; and/or HY2, which converts biliverdin to phytochromobilin.

In certain embodiments, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used. Suitable selection genes can include, for example, genes coding for ampicillin and/or tetracycline resistance, which enables cells transformed with these vectors to grow in the presence of these antibiotics.

In one aspect of the present invention, a nucleic acid encoding a far-red CBCR of the invention is introduced into a cell, either alone or in combination with a vector. By “introduced into,” it is meant that the nucleic acids enter the cells in a manner suitable for subsequent integration, amplification, and/or expression of the nucleic acid. The method of introduction or transfection is largely dictated by the targeted cell type. Exemplary methods include CaPO₄ precipitation, liposome fusion, LIPOFECTIN®, electroporation, heat shock, viral infection, and the like.

In some embodiments, prokaryotes are used as host cells for the initial cloning steps of the present invention. Other host cells include, but are not limited to, eukaryotic (e.g., mammalian, plant and insect cells), or prokaryotic (bacterial) cells. Exemplary host cells include, but are not limited to, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Sf9 insect cells, and CHO cells. They are particularly useful for rapid production of large amounts of DNA, for screening many mutants simultaneously, and for DNA sequencing of the mutants generated. Suitable prokaryotic host cells include E. coli K12 strain 94 (ATCC No. 31,446), E. coli strain W3110 (ATCC No. 27,325), E. coli K12 strain DG116 (ATCC No. 53,606), E. coli X1776 (ATCC No. 31,537), and E. coli B; and other strains of E. coli, such as LMG194, HB101, JM101, NM522, NM538, and NM539. Many other species and genera of prokaryotes including bacilli such as Bacillus subtilis, other enterobacteriaceae such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species can all be used as hosts. Prokaryotic host cells or other host cells with rigid cell walls are typically transformed using the calcium chloride method as described in Sambrook et al., supra. Alternatively, electroporation can be used for transformation of these cells. Prokaryote transformation techniques are set forth in, for example Dower, in Genetic Engineering, Principles and Methods 12:275-296 (Plenum Publishing Corp., 1990); Hanahan et al., Meth. Enzymol., 204:63, 1991. Plasmids typically used for transformation of E. coli include pBR322, pUCI8, pUCI9, pUCI18, pUC119, and Bluescript M13, all of which are described in sections 1.12-1.20 of Sambrook et al., supra. However, many new expression vectors are available as well.

Accordingly, some embodiments of the invention provide a host cell comprising a far-red CBCR nucleic acid, expression cassette, or vector, as described herein. In some embodiments, the far-red CBCR nucleic acid, expression cassette, or vector in the host cell encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOS: 1-28 (e.g., SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7).

In some embodiments, the far-red CBCR protein fusion constructs of the present invention are produced by culturing a host cell transformed with an expression vector containing a nucleic acid encoding the far-red CBCR, under the appropriate conditions to induce or cause expression of the far-red CBCR. Methods of culturing transformed host cells under conditions suitable for protein expression are well known in the art (see, e.g., Sambrook et al., supra). Suitable host cells for production of the far-red CBCR protein fusion constructs from T7 promoter-containing plasmid vectors include E. coli strain BL21 (DE3) and related lysogens (see, e.g., U.S. Pat. No. 5,693,489). Following expression, a far-red CBCR protein fusion construct can be harvested and isolated.

IV. Methods

The far-red CBCR protein fusion constructs of the invention are useful as fluorescent markers in the many ways fluorescent markers already are used. This includes, for example, coupling far-red CBCR domains to antibodies, nucleic acids, or other receptors for use in detection assays, such as immunoassays or hybridization assays. Such constructs are particularly useful in applications involving the monitoring of gene expression and protein localization. Far-red CBCRs are ideal for such applications as they are readily detectable, can be detected on irradiation using standard long-wave light sources; offer the possibility of real-time detection in vivo; do not require introduction of a substrate to produce a signal; and allow manageable protein fusions due to their relatively small size and monomeric nature.

Accordingly, another aspect of the invention provides a method for detecting a cellular component, the method comprising:

-   -   providing a protein fusion construct in a sample, the fusion         construct comprising a far-red CBCR domain and a heterologous         domain detecting a cellular component;     -   exposing the protein fusion construct to far-red light or         near-IR light, wherein the exposing causes fluorescence of the         far-red CBCR domain; and     -   detecting the fluorescence of the far-red CBCR domain,     -   thereby detecting the cellular component.

In some embodiments, the sample is a cell or tissue and wherein providing the protein fusion construct comprises expressing the protein fusion construct in the cell or tissue.

As a non-limiting example, the invention provides for antibodies or antibody fragments to which the far-red CBCR labels of the invention are joined. The antibodies are capable of specifically binding to the antigen to which they are directed. Detection of the presence, absence, or particular amount of far-red/near-IR fluorescence provides an indication of presence, absence, or amount of analyte to which the antibody is directed.

Similarly, far-red CBCR label labeled antibodies, or other ligands, can be used in immunohistochemical applications. In this context, far-red CBCR antibody constructs are used to probe cells, tissues, and sections thereof. When the subject sample is contacted with the antibody construct, the antibody binds and localizes to specific regions of the sample in which the target molecule (the molecule or moiety recognized by the antibody) is located. Localization and/or quantification of the far-red/near-IR fluorescence provides information concerning the location and/or quantity of the target molecule in the sample. One of skill in the art will appreciate that the far-red CBCR constructs are also well suited for in situ and in vivo labeling of molecules, cells, and cellular components.

As another non-limiting example, the far-red CBCR constructs can be used for probing protein-protein interactions. In certain embodiments, two apoprotein cDNA constructs are used. The first construct will encode an apoprotein species whose assembly with a given bilin emits at a well-defined wavelength (donor). The second construct will encode an apoprotein species whose assembly with the same, or different, bilin produces a fluorescent species that both absorbs and emits light at longer wavelengths (acceptor). Protein-protein interaction between two proteins of interest (e.g., protein X and protein Y) is identified following their co-expression as translational fusions with apoprotein in constructs 1 (donor) and 2 (acceptor) using fluorescence energy transfer from the shorter wavelength-absorbing donor species to the longer wavelength-absorbing acceptor species.

In another application, chimeric apoprotein-protein X cDNA (where protein X is any protein of interest) are expressed in transgenic eukaryotes (yeast, plants, Drosophila, etc.) in order to study the subcellular localization of protein X in situ. Following feeding of exogenous bilin or engineering bilin biosynthesis in target tissues, subcellular localization can be performed using fluorescence microscopy (e.g., laser confocal microscopy).

In some embodiments, the far-red CBCR protein fusion constructs of the invention are used as in vitro or in vivo labels in a manner analogous to the use of Green Fluorescent Protein (GFP). This typically involves transfecting a cell with a nucleic acid encoding an apoprotein in such an manner that the cell expresses the apoprotein (e.g., the nucleic acid is a component of an expression cassette). When the apoprotein is provided with the appropriate bilin, supplied either exogenously or produced endogenously, the far-red CBCR label (fluorescent adduct) self assembles and thereby produces a fluorescent marker. Uses of such markers are well known to those of skill in the art (see, e.g., U.S. Pat. No. 5,491,084 which describes uses of GFP).

In a related aspect, the invention provides a method for imaging a biological structure in a subject, the method comprising:

-   -   providing a protein fusion construct in or near the biological         structure, the fusion construct comprising a far-red CBCR domain         and a heterologous domain;     -   exposing the protein fusion construct to far-red light or         near-IR light, wherein the exposing causes absorbance by, or         release of an acoustic signal or fluorescence from, the far-red         CBCR domain;     -   detecting the absorbance, acoustic signal, or fluorescence of         the far-red CBCR domain; and     -   constructing an image of the biological structure;     -   thereby imaging the biological structure.

In some embodiments, the biological structure is selected from the group consisting of a tissue, an organ, or a tumor. In the imaging methods, the far-red CBCR domain can be used in the manner of other infrared dyes (e.g., phthalocyanine dyes, naphthalocyanine dyes, polymethine dyes, quinone dyes, and azo dyes) as described, for example, in U.S. Pat. Nos. 6,083,485; 9,089,603; 8,463,365; and 9,201,014. Examples of imaging techniques include, but are not limited to, optical coherence tomography and photoacoustic tomography (see, Matcher, Methods Mol Biol. 2011; 695: 261-80; Yao et al. Nat Methods. 2016 (1):67-73).

In some embodiments, the protein fusion construct is expressed in a cell or organism prior to imaging. Expression can be transient or permanent. With respect to expression in organisms, expression can be limited to specific cell types, tissue types, or developmental stages, or the protein fusion construct can be expressed throughout the entire organism. Suitable cells include bacterial cells, insect cells, fungal cells, yeast cells, plant cells, animal cells, mammalian cells, human cells, cancer cells, and stem cells.

In particular embodiments, the organism is a transgenic animal. Non-limiting examples of animals suitable for transgenesis include mice, rats, guinea pigs, rabbits, livestock (e.g., cattle, sheep, chickens, goats, pigs, salmon, trout, carp, catfish, silkworms), zebrafish, tilapia, frogs, and fruit flies. Methods for introducing recombinant nucleic acids for achieving transgenesis and inducing expression are described herein and will be known to one of skill in the art.

In another aspect, the invention provides a method for modulating a cellular process, the method comprising:

-   -   expressing a protein fusion construct in a cell, the fusion         construct comprising a far-red CBCR domain and a heterologous         signaling domain;     -   exposing the protein fusion construct to far-red light or         near-IR light;     -   wherein the exposing increases or decreases the activity of the         heterologous signaling domain, thereby modulating the cellular         process.

In some embodiments, the heterologous signaling domain is selected from the group consisting of a kinase polypeptide, a phosphatase polypeptide, a phosphodiesterase polypeptide, a nucleotide cyclase polypeptide, a protease, a phopholipase, a G-protein polypeptide, and a channel protein polypeptide. Methods for control of output domains (e.g., adenylyl cyclases, phosphodiesterases, caspases, etc.) using light-activated sensors (e.g., phytochrome domains, bacteriophytochrome domains, and the like) can be applied to the methods for controlling cellular processes. See, for example, U.S. Pat. Nos. 8,828,658 and 8,835,399.

Genes encoding the far-red CBCR protein fusion constructs can be introduced into live animals, plants or microbes, where their activities can be turned on (or off) by far-red/near-IR light, controlled by the duration and/or intensity of light, and turned off (or on) by light of a different wavelength than the activating light. Diverse cellular processes can be regulated with high spatial and temporal precision in a nontoxic manner, often using external light sources. For example, far-red CBCR protein fusion constructs possessing nucleotidyl cyclase, protein kinase, protease, DNA-binding and RNA-binding activities can be used to control metabolic enzymes, signal transduction, cell apoptosis, proliferation, adhesion, differentiation and other processes.

In some embodiments, the protein fusion construct is expressed in a transgenic animal. Non-limiting examples of animals suitable for transgenesis include mice, rats, guinea pigs, rabbits, livestock (e.g., cattle, sheep, chickens, goats, pigs, salmon, trout, carp, catfish, silkworms), zebrafish, tilapia, frogs, and fruit flies.

In other embodiments, the protein fusion construct is expressed in a transgenic plant. Non-limiting examples of plants suitable for transgenesis include tobacco, maize, rice, potato, apple, tomato, wheat, sunflower, soybean, carrot, radish, spinach, and alfalfa.

In some other embodiments, the protein fusion construct is expressed in a transgenic microbe. Non-limiting examples of microbes suitable for transgenesis include BL21 E. coli, DE3 strain E. coli, E. coli M15, DH5α, DH10β, HB101, T7 Express Competent E. coli (NEB), B. subtilis cells, Pseudomonas fluorescens cells, Chlamydomonas reinhardtii cells, Synechococcus elongatus cells, Pyrococcus furiosus, Metallosphera sedula, Thermococcus litoralis, Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Pyrococcus abyssi, Sulfolobus solfataricus, Pyrococcus woesei, Sulfolobus shibatae, and variants thereof.

A far-red/near-IR light-activated executor (effector) caspase can be introduced into tumors (or other kinds of disease-causing cells, e.g., cells carrying viruses) to induce an apoptotic cell death pathway, thus providing a noninvasive gene therapy of cancer (or viral diseases). Human cells expressing hormones (e.g., insulin) can be regulated by far-red CBCR protein fusion constructs (e.g., due to the light-regulated gene expression or hormone-synthesizing activity) and can be used to treat hormone deficiencies (e.g., diabetes). Far-red CBCR protein fusion constructs can be used to photoactivate immune cells at desired locations (e.g., tumor or infection sites). Far-red CBCR protein fusion constructs can also be used to convert prodrugs into active drugs in irradiated tissues and/or organs. Far-red CBCR protein fusion constructs expressed in bacteria (e.g., E. coli or Lactobacillus) that belong to normal human or animal microflora can be used to photoactivate organ-localized (e.g., colon, vagina) synthesis of bacteriophages, antibiotics, and other drugs to target pathogenic microorganisms, polyps and tumors or to produce probiotics. Some far-red CBCR protein fusion constructs can be used as protein-based drugs directly (e.g., by light-activated binding and control of cellular receptors).

The output module can be selected from enzymes and other proteins that have a desired biological activity, e.g., enzymatic activity, or ability to bind DNA, RNA or other proteins. In some embodiments, the output modules can include protein kinases, proteases (including caspases), nucleotidyl cyclases, nucleases (including recombinases), DNA-binding and RNA-binding protein modules, and others that are activated by homodimerization.

In certain instances, the far-red CBCR protein fusion constructs can be activated or their activity can be enhanced by the application of light of an activating wavelength. They can be inactivated, or their activity can be reduced by the absence of light or by the application of light of an inactivating wavelength. Some far-red CBCR protein fusion constructs can be active or show enhanced activity in the dark or reduced light, and be inactivated or show reduced activity when light of an inactivating wavelength is applied. The “absence of light” can mean the absence of all light (i.e., darkness), or can mean the absence of light in a selected wavelength range that causes a change in the conformation of the CBCR protein module.

Thus, in some embodiments the desired activity is increased by the application of far-red/near-IR light of a selected wavelength. In some embodiments, the desired activity is decreased by the application of far-red/near-IR light of a selected wavelength. In some embodiments, the desired activity is gradually decreased or gradually increased by ceasing to apply far-red/near-IR light of a selected wavelength. In some embodiments, the desired activity is immediately increased or decreased by the application of NIR light of a selected wavelength. Suitable selected wavelengths are determined by the spectral properties of the CBCR domain.

It is to be understood that the terms “active” and “inactive” in the foregoing explanation are relative and include complete activity of the protein to complete inactivity of the protein (complete “on/off” modes) as well as relative activity or inactivity of the proteins, i.e., the fusion protein constructs can have high activation ratios, low activation ratios, or activation ratios between high and low. In some embodiments, the fusion protein constructs can be controlled by light so as to have relatively high ratios (e.g., about 2:1 or greater, about 5:1, or about 10:1 or greater) of activity to inactivity or of inactivity to activity under the control of light of appropriate wavelengths.

V. Examples

The present invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.

Disclosed herein is the discovery of CBCRs with peak absorption in the far-red window. Phylogenetic analysis identified a small CBCR cluster related to green/red CBCRs but exhibiting differences in a key sequence motif. In vitro characterization of five such proteins after recombinant expression in Escherichia coli revealed three with a conserved far-red/orange photocycle and two with a conserved far-red/red photocycle. The far-red-absorbing chromophore adopts a reversed chemical configuration relative to that of the far-red-absorbing P_(fr) state of phytochromes and is a covalent PCB adduct similar to those of many other CBCR lineages (FIG. 1), but with a remarkable red shift of 70-80 nm in the native protein. Moreover, some far-red/orange CBCRs exhibit detectable far-red and near-infrared fluorescence. These studies establish far-red CBCRs as promising compounds for diverse applications in live cells and provide new insight into detection of far-red and near-IR light by bilin chromophores.

Example 1. Materials and Methods for Discovery and Characterization of Far-Red and Near-IR CBCRs

Bioinformatics. CBCR sequences were identified using BLAST searches[56] against the Genbank and DOE-IMG databases. Locus tags from DOE-IMG are reported in Tables 1 and 3. All phylogenies were calculated using maximum-likelihood methods with structural information in PhyML-structure.[57] To generate the final alignment used for calculating the CBCR phylogeny shown in FIG. 2, new CBCR sequences were manually added to a pre-existing alignment.[54] The resulting alignment was pruned, and the region encompassing the Asp-motif was manually adjusted to optimize conservation of hydrophobic residues. Key regions are presented in FIG. 3. Structural information was projected onto the sequence alignment using an in-house script as described[54] using CBCR crystal structures for TePixJ and AnPixJ (PDB accession codes 3W2Z, 4FOF, and 4GLQ).[58, 59] TePixJ and AnPixJ themselves are not associated with histidine kinases and hence were removed for final phylogeny calculation, because the encoding of the structural information in PhyML-structure is not tied to the individual sequences. [57] C-terminal His kinase regions were initially aligned to sequences from histidine kinases for which crystal structures were available (PDB accession codes: 2C2A, 3DGE, 4U7N, 4U70, 3D36, 4R39) [60-63] using MUSCLE.[64] The resulting alignment was adjusted manually and structural information was added to the alignment using the in-house script as described above to yield the alignment used for calculating the maximum-likelihood phylogeny shown in FIG. 2. Key regions are presented in FIG. 4. Sequences for the crystal structures were again removed prior to calculation of the phylogeny, because those sequences were not associated with His kinase domains and hence would not be matched in the ‘tanglegram’ representation of FIG. 2.

Both phylogenies were calculated in PhyML-structure using the six-matrix EX_EHO model in partitioning mode, using the LG substitution matrix for positions with no structural information. [57] Maximum likelihood estimates were used for the proportion of invariable sites and for the distribution of the gamma shape parameter, with four substitution categories and optimization of tree topology, branch length, and rate parameters. The resulting tree was processed using FigTree (available online) and graphics editing software.

Cloning, Expression, and Purification of CBCRs.

Anacy_4718g3 (amino acids 1274-1466 of the Anacy_4718 locus in Anabaena cylindrica PCC 7122), Anacy_2551g3 (amino acids 835-1026) of Anacy_2551), and Oscil6304_4080 (amino acids 341-515 of Oscil6304_4080 in Oscillatoria acuminata PCC 6304) were cloned from genomic DNA prepared from Anabaena sp. PCC 7938 and Oscillatoria acuminata PCC 6304 using PCR with appropriate primers and with addition of one to two amino acids at the N terminus to create a start codon with an NcoI restriction site. For Anabaena sp. PCC 7938, amplified DNA sequences were identical to those of A. cylindrica PCC 7122.

Cyan7822_4053g2 (amino acids 903-1091 of Cyan7822_4053 in Cyanothece sp. PCC 7822), Nos7524_4790 (amino acids 932-1105 of Nos7524_4790 in Nostoc sp. PCC 7524), Sta7437_1656 (amino acids 696-871 of Sta7437_1656 from Staniera cyanosphaera PCC 7437), and WP_016871037 (amino acids 1246-1419 of UYKDRAFT_01008 from Fischerella thermalis PCC 7521) were obtained as synthetic genes (Genscript, Piscataway, N.J.) codon-optimized for expression in E. coli.

Anacy_4718g3, Cyan7822_4053g2, Nos7524_4790, and Oscil6304_4080 were cloned into pBAD-Cph1-CBD[65] using unique NcoI and SmaI sites, generating in-frame fusions to a C-terminal intein-CBD tag. Expression in E. coli strain LMG194 with co-production of PCB using pPL-PCB followed published procedures.[66] Co-production of PΦB and PEB was performed in the same way, but used pPL-PΦB and pAT-PebS, respectively.[22, 45] Proteins were purified on chitin resin (NEB) as previously described, with final dialysis into TKKG buffer (25 mM TES-KOH pH 7.8, 100 mM KCl, 10% (v/v) glycerol). [54, 65]

Anacy_2551g3, Anacy_4718g3, Sta7437_1656, and WP_016871037 were cloned into pET28-RcaE[17] using unique NcoI and BamHI sites, thereby cloning each CBCR as an in-frame fusion to a C-terminal His tag. His-tagged proteins were expressed in E. coli strain C41[67] with co-production of PCB using pKT271[68] and were purified on purified on His-bind Ni²⁺-NTA resin (Novagen) using an imidazole gradient.[17, 42] His-tagged proteins were dialyzed into 20 mM sodium phosphate (pH 7.5), 50 mM NaCl, 10% (v/v) glycerol and 1 mM EDTA.

Purified proteins were analyzed by SDS-PAGE using standard procedures and apparatus (Bio-Rad) followed by semi-dry transfer to PVDF membranes, staining with amido black for visualizing total protein, and zinc blotting (FIG. 5).[69]

Spectroscopic Characterization of CBCRs.

Absorption spectra were acquired on a Cary 50 spectrophotometer at 25° C. Photoconversion was triggered in the absorption cuvette using 728 nm LEDs (Sanyo) or using a xenon source equipped with band-pass interference filters (400±35 nm, 550±35 nm, 600±20 nm, 650±20 nm).[65] For WP_016871037, a red laser pointer (632.8 nm, 2 mW) was used as well. Fluorescence spectra were acquired on a QM-6/2005SE fluorimeter equipped with red-enhanced photomultiplier tubes (Photon Technology International). For denaturation assays, a 100 μl aliquot of protein was added to 1 ml of 7 M guanidinium chloride/1% HCl (v/v). Denatured samples were illuminated using the xenon lamp equipped with a 320 nm long-pass filter, and extinction coefficients were estimated from the denatured spectra as described previously[51] using the known extinction cofficients for PCB under acid denaturation conditions.[70] The fluorescence quantum yield of Anacy_2551g3 was estimated using the ratio method with Alexa 750 (Thermo Fisher) as the reference standard. [22]

Example 2. Discovery of Conserved Far-Red/Orange and Far-Red/Red CBCR Lineages

Previous phylogenetic analyses clustered CBCR domains Anacy_4718g3 and Anacy_2551g3 from the filamentous cyanobacterium Anabaena cylindrica PCC 7122 with green/red CBCRs.[54] These sequences diverge from those of canonical green/red CBCRs, particularly in the Asp-motif region associated with spectral tuning in many CBCR lineages.[17, 43, 54, 59, 71] These sequences were therefore assessed to determine if they might be part of a previously uncharacterized CBCR lineage by using them as queries in BLAST [56] searches. This approach identified additional CBCR sequences containing similar variant Asp-motifs (Table 1).

TABLE 1 Accession information for far-red CBCRs Sequence Domain architecture DOE-IMG accession Organism WP008316973g2 HAMP-PAS₁₀-CBCR-CBCR- LEP6406DRAFT_2712 Leptolyngbya PCC HK-REC 6406 C56DFT2_02270g2 PAS₂-CBCR-CBCR-HK-REC cya56DRAFT2_02270 Planktothrix agardhii NIVA-CYA 56/3 c407DFT_01196g2 PAS₄-CBCR-CBCR-HK-REC Ga0039498_104087 Planktothrix rubescens NIVA- CYA 407 Ga0039499_10213 PAS₄-CBCR-CBCR-HK-REC Ga0039499_10213 Planktothrix prolifica NIVA-CYA 406 WP017296986g2 PAS₇-CBCR-CBCR-HK-REC Lepto7104DRAFT_1307 Leptolyngbya PCC 7104 (Nodosilinea nodulosa) Cy7822_4053g2 CBS₂-PAS₃-CBCR-CBCR- Cyan7822_4053 Cyanothece sp. PCC HK-REC 7822 C6303_3693g3 CBS₄-GAF-CBCR-HK-REC Cal6303_3693 Calothrix parietina PCC 6303 L6406D_1154g2 CBS₄-GAF-PAS₁₀-CBCR- LEP6406DRAFT_1154 Leptolyngbya PCC CBCR-HK-REC 6406 Anacy_2551g3 CBS₄-CBCR-PAS-CBCR- Anacy_2551 Anabaena cylindrica CBCR-HK PCC 7122, PCC 7938 WP028089844g3 CBS₃-CBCR-PAS₃-CBCR- 131C_1565 Anabaena circinalis CBCR-HK AWQC131C 310F_3509 CBS₃-CBCR-PAS₃-CBCR- 310F_3509 Anabaena circinalis CBCR-HK AWQC310F WP006632756g3 CBS₄-CBCR-PAS₁₁-CBCR- MicvaDRAFT_3059 Microcoleus CBCR-HK vaginatus FGP-2 N7104D_1016g3 CBS₂-CBCR-PAS₇-CBCR- Lepto7104DRAFT_1016 Leptolyngbya PCC CBCR-HK-REC 7104 (Nodosilinea nodulosa) fdiDRAFT29700 CBS₃-CBCR-PAS₂-CBCR- fdiDRAFT29700 Tolypothrix sp. PCC CBCR-HK-REC 7601 Anacy_4718g3 CBS₄-CBCR-PAS₃-CBCR- Anacy_4718 Anabaena cylindrica CBCR-HK-REC PCC 7122, PCC 7938 WP009627289g3 CBS₂-CBCR-PAS₃-CBCR- Pse7429DRAFT_2072 Pseudanabaena sp. CBCR-HK-REC PCC 7429 Os7112_5903g3 CBS₄-CBCR-PAS₁₁-CBCR- Osc7112_5903 Oscillatoria nigro- CBCR-HK-REC viridis PCC 7112 WP_033374293 CBS₄-CBCR-PAS₅-GAF- Spi9445_1327 Spirulina subsalsa CBCR-HK-REC PCC 9445 Cy7425_1390g3 CBS-CBCR-PAS-GAF-PAS₄- Cyan7425_1390 Cyanothece sp. PCC CBCR-CBCR-HK-REC 7425 Sta7437_1656 XXX-HAMP-PAS₂-CBCR- Sta7437_1656 Staniera cyano- PAS-HK sphaera PCC 7437

In Table 1, abbreviations for domains are as follows. CBS: cystathionine-β-synthase; CBCR: cyanobacteriochrome domain; PAS: Per-ARNT-Sim; GAF: cGMP-specific phosphodiesterases, cyanobacterial adenylate cyclases, and formate hydrogen lyase transcription activator FhlA; HAMP: Histidine kinases, Adenylate cyclases, Methyl-accepting proteins and Phosphatases; HK, histidine kinase bidomain; REC, response regulator receiver domain. Numbers indicate multiple domains of a given type in tandem, and CBCRs belonging to the far-red cluster are indicated in bold.

These CBCRs could readily be aligned with those of known green/red CBCRs (FIG. 3). Maximum-likelihood phylogenetic analysis demonstrated that these sequences, including Anacy_4718g3 and Anacy_2551g3, formed part of a distinct cluster (FIG. 2). CBCR domains in this cluster were part of larger signaling molecules with C-terminal histidine kinases (Table 1 & FIG. 4) and possessed diverse full-length domain architectures associated with at least three different subfamilies of C-terminal histidine kinase “output” domains (FIG. 2). By contrast, coherent CBCR/kinase pairings were observed for the green/red CBCRs RcaE and CcaS associated with CCA (FIG. 2). Taken together, these studies establish Anacy_4718g3 and Anacy_2551g3 as members of a new subfamily of CBCR photosensors associated with evolutionarily diverged signaling proteins.

Next, Anacy_4718g3 was characterized in vitro after recombinant expression in E. coli engineered to produce PCB.[66] This protein exhibited photoconversion between far-red-absorbing and orange-absorbing states exhibiting peak absorption at 740 nm and 590 nm, respectively (FIG. 6A & Table 2). Similar photocycles were observed for two constructs with different affinity tags (FIG. 7A-B), indicating that the different reagents employed in purifying His-tagged proteins or intein-CBD fusion proteins did not affect the far-red/orange photocycle. Slow dark reversion from the orange-absorbing state to the far-red-absorbing state was observed, indicating that the far-red-absorbing state is dark-adapted and the orange-absorbing state is the photoproduct (FIG. 7C). Anacy_2551g3 exhibited a similar photocycle (FIG. 6B), with the far-red-absorbing maximum at a slightly shorter wavelength (728 nm: Table 1). A third member of this cluster, Cyan7822_4053g2 from the unicellular cyanobacterium Cyanothece sp. PCC 7822, exhibited almost identical behavior to Anacy_2551g3 (FIG. 6C & FIG. 7D; Table 2). These results establish the existence of a cluster of CBCRs related to green/red CBCRs but exhibiting conserved far-red/orange photocycles.

TABLE 2 Spectral properties of CBCRs Protein Construct Bilin 15Z λ_(max) 15E λ_(max) SAR Anacy_4718g3 intein-CBD PCB 740 590 0.16 Anacy_4718g3 intein-CBD PΦB 752 590 0.12 Anacy_4718g3 intein-CBD PEB 610 — 0.37 Anacy_4718g3 intein-CBD BV — — <0.02 Anacy_4718g3 poly-His PCB 740 590 0.17 Anacy_2551g3 poly-His PCB 728 588 0.13 Cyan7822_4053g2 poly-His PCB 728 590 0.07 Sta7437_1656 poly-His PCB 728 642 0.35 WP_016871037 poly-His PCB 726 640 0.34 Oscil_6304_4080 intein-CBD PCB 544 — 0.63 Nos7524_4790 intein-CBD PCB 418 658 0.17

In Table 2, all peak wavelengths are reported in nm. Specific Absorbance Ratio (SAR) was calculated as the ratio of the peak absorbance of the longest-wavelength chromophore band for the 15Z photostate to the peak absorbance of the aromatic amino acid band at 280 nm and serves as a relative measure of chromophore loading for a given protein/chromophore combination. The 15Z value for Anacy_4718g3 is for the dark-adapted state. The SAR value for WP_016871037 was estimated after subtraction of 15E signals

Phylogenetic analysis tentatively placed far-red/orange CBCRs as part of a larger lineage that is sister to the CCA photoreceptors CcaS and RcaE (FIG. 2). Within this lineage, one branch of CBCRs includes proteins with Asp-motifs very similar to those of CcaS and RcaE, such as PlpA from Synechocystis sp. PCC 6803[72] and Oscil6304_4080 from Oscillatoria acuminata PCC 6304 (FIG. 3). The other branch includes both the far-red/orange CBCRs and other sequences. Some of these other sequences have Cys residues in or near the Asp-motif (FIG. 3), including Nos7524_4790 and Sta7437_1656. Such Cys residues can form a second covalent linkage to the chromophore, resulting in absorption of blue to ultraviolet light.[43, 45, 73] Additional CBCRs were therefore characterized to explore the transition from green/red photocycles to far-red/orange photocycles.

Oscil6304_4080 exhibited a dark-adapted state with peak absorption in the green region of the spectrum (FIG. 8A). Illumination with green light (550±35 nm) produced only minimal photoconversion. The peak wavelength and lineshape of the green-absorbing state were similar to those of the green-absorbing states previously reported for the CCA regulator RcaE from Fremyella diplosiphon [17] and the green/blue CBCR Oscil6304_4336g2 from O. acuminata (FIG. 8B). Comparison of the normalized difference spectra for Oscil6304_4080 and RcaE shows similar depletion of the green state, but Oscil6304_4080 fails to form the red-absorbing photoproduct state (FIG. 8C). Nos7524_4790 exhibited photoconversion between two photostates with peak absorption in the blue and red regions of the visible spectrum (FIG. 8D & Table 2). The red-absorbing state was again similar to that of RcaE (FIG. 8E). Neither Oscil6304_4080 nor Nos7254_4790 exhibited detectable species with far-red absorption.

Sta7437_1656 exhibited photoconversion between states with far-red and red peak absorption (FIG. 6D). The far-red state was very similar to that of Anacy_2551g3, whereas the red-absorbing state was blue-shifted relative to those of RcaE and Nos7524_4790 (FIG. 7E-F). As purified, WP_016871037 exhibited a mix of red- and far-red-absorbing species (FIG. 6E), with ready conversion of the far-red state to the red state but poor reversibility even after laser illumination (FIG. 7G). The far-red/red photochemical difference spectrum for WP_016871037 was similar to that of Sta7437_1656 (FIG. 7H). Sta7437_1656 and WP_016871037 are part of a small cluster of CBCR domains associated with clustered histidine kinase output domains (FIG. 2) but having diverse domain architectures (Table 3). These results implicate a second cluster of far-red CBCRs with far-red/red photocycles (FIG. 2).

TABLE 3 Accession information for far-red/red CBCRs Sequence Domain architecture DOE-IMG accession Organism Sta7437_1656 CACHE-HAMP-PAS₂-CBCR-PAS- Sta7437_1656 Stanieria cyanosphaera PCC HK 7437 WP_016871037 TPR-GAF-PAS₇-CBCR-PAS₃-HK UYKDRAFT_01008 Fischerella thermalis PCC 7521 WP_026722600 GAF-PAS₆-CBCR-PAS₂-HK Fis9431DRAFT_3998 Fischerella sp. PCC 9431 WP_017309337 GAF-PAS₄-CBCR-PAS₂-HK PCC9339DRAFT_00524 Fischerella sp. PCC 9339 WP_016878855 REC-PAS₆-CBCR-PAS-HK UYEDRAFT_06529 Chlorogloeopsis fritschii PCC 9212 WP016873240 CACHE-HAMP-PAS₄-GAF-CBCR- UYEDRAFT_00976 Chlorogloeopsis fritschii PCC PAS-HK 9212

In Table 3, abbreviations for domains follow those of Table S 1. Additonal domains: TPR, tetratricopeptide repeat domain; CACHE: Ca²⁺ channels, chemotaxis protein.

Spatial Separation of Photoconversion and Spectral Tuning in the Far-Red-Absorbing Chromophore.

The far-red-absorbing states of these newly described CBCRs exhibited remarkable red shifts relative to previously known CBCRs (ca. 650-710 nm)[17, 42, 46, 48, 49, 51-53]. Therefore, an acid denaturation assay[73-77] was used to examine chromophore structure in far-red CBCRs. In this assay (FIG. 9A), samples in either photostate are denatured by dilution into concentrated guanidinium chloride. In the absence of native protein structure, 15E bilins can be unidirectionally photoconverted to the 15Z configuration by white light, allowing assignment of the chemical configuration of the photostates. Different bilins have characteristic spectra under denaturing conditions, with different peak wavelengths and with different relative intensities for the long- and short-wavelength chromophore absorption bands in the ultraviolet to visible spectrum (FIG. 9B). Although it is possible for labile structural changes to be lost upon denaturation, this assay can provide tentative identification of bilin species. [45, 47, 73]

Acid denaturation of Anacy_4718g3 resulted in the expected spectral changes and also revealed the presence of a porphyrin side population. Photoconversion of denatured samples established the far-red-absorbing state as having the 15Z configuration and the orange-absorbing state as having the 15E configuration (FIG. 10A-B). Denaturation analysis of Anacy_2551g3 and Cyan7822_4053g2 confirmed that the far-red-absorbing states of all three far-red/orange CBCRs adopted the 15Z configuration (FIG. 11A-B), albeit with varying amounts of porphyrin. The far-red/red CBCR Sta7437_1656 exhibited a 15Z far-red-absorbing photostate and 15E red-absorbing photoproduct, with notably less contaminating porphyrin (FIG. 10C-D). Nos7524_4790 exhibited a similar red-absorbing 15E photoproduct, in this case with a blue-absorbing 15Z dark state (FIG. 8F). As purified, WP_016871037 exhibited both far-red- and red-absorbing species, confirmed as a mix of 15Z and 15E bilin by acid denaturation (FIG. 11C). Photoconversion of this protein with far-red light resulted in incomplete formation of 15E bilin (FIG. 11D), assigning the red-absorbing state as the 15E photoproduct. The presence of 15E photoproduct in this preparation arose due to the combination of light exposure during purification and poor reverse photoconversion in this protein (see above). Estimation of the extent of photoconversion in both samples by comparison to reference spectra[17] allowed for the subtraction of a scaled photoproduct spectrum from the initial spectrum, resulting in a spectrum similar to that of Sta7437_1656 in the far-red-absorbing photostate (FIG. 6E). These results demonstrate that far-red CBCRs exhibit a conserved 15Z photostate with peak absorption in the far-red and with blue-shifted 15E photoproducts absorbing orange or red light. Far-red CBCRs thus are reversed relative to phytochromes, in which the red-absorbing 15Z Pr state is blue-shifted relative to the far-red-absorbing 15E P_(fr) state. [34, 78, 79]

The photochemical difference spectrum for denatured Anacy_4718g3 was superimposable on that of the red/green CBCR NpR6012g4 (FIG. 12A). Recent characterization of NpR6012g4 using solution NMR spectroscopy confirmed the presence of a covalent PCB adduct in both photostates, with intramolecular nuclear Overhauser effect cross-peaks confirming photoisomerization at the 15,16-double bond.[39, 80] The peak wavelength and relative bilin band intensities of denatured 15Z Anacy_4718g3 in the far-red state were also consistent with a 15Z covalent PCB adduct (FIG. 12B). Therefore, both photostates of Anacy_4718g3 contain a covalent PCB adduct, despite the fact that 15Z PCB adducts typically absorb at 530-670 nm rather than 720-740 nm.[17, 22, 34, 42, 44, 46, 51, 52, 55, 81-83]

Example 3. Chromophore Structure in the Far-Red State

Next, Anacy_4718g3 was characterized after co-expression with other bilins (FIG. 1). BV did not bind efficiently, but Anacy_4718g3 adducts with phytochromobilin (PΦB) and phycoerythrobilin (PEB) were obtained (FIG. 13A). Anacy_4718g3-PΦB exhibited a red shift of the far-red-absorbing state to 752 nm relative to Anacy_4718g3-PCB, but photoconversion with far-red light resulted in formation of a photoproduct having similar photoproduct effect peak absorption to that of Anacy_4718g3-PCB (FIGS. 13B-C & Table 2). Denaturation analysis confirmed the presence of PΦB (FIG. 14D). The 18-ethyl moiety of PCB is instead an 18-vinyl in PΦB (FIG. 1), providing one extra double bond in the conjugated π-electron system of PΦB. PΦB adducts of biliproteins are thus red-shifted relative to PCB adducts.[22, 47, 51, 81, 84] Indeed, the red shift observed for Anacy_4718g3-PΦB relative to Anacy_4718g3-PCB is consistent with those observed for a broad range of CBCRs upon introduction of the 18-vinyl moiety (FIG. 15A). By contrast, the extinction coefficient of the PCB adduct did not follow the general correlation between peak wavelength and extinction coefficient observed for PCB and phycoviolobilin adducts of most other CBCRs (FIG. 15B & Table 4).

TABLE 4 Estimated extinction coefficients for far-red CBCRs in this study. Protein 15Z ε (M⁻¹ cm⁻¹) Anacy_4718g3 (intein-CBD) 40,800 Anacy_4718g3 (His tag) 44,400 Anacy_2551g3 (His tag) 49,200 Cyan7822_4053g2 (intein-CBD) 39,900 Sta7437_1656 (His tag) 43,600

In Table 4, all values are for PCB adducts. Comparison of values for different Anacy_4718g3 constructs suggests an error range of ±10%. WP_016871037 was excluded because a native 15Z spectrum could not be obtained in the absence of 15E bilin.

PEB differs from PCB and PΦB in having a saturated 15,16-bond that results in loss of conjugation from the bilin D-ring (FIG. 1). PEB adducts of phytochromes are therefore blue-shifted and cannot undergo photoconversion.[20] Anacy_4718g3-PEB exhibited peak absorption at 610 nm (FIG. 13A), blue-shifted relative to the PCB adduct, and was also photoinactive (FIG. 13C). The absorption maximum of Anacy4718g3-PEB is red-shifted ca. 50 nm relative to PEB adducts of other CBCRs (550-560 nm),[50] but denaturation analysis confirmed the presence of PEB (FIG. 13E). A linear relationship was observed between the peak wavelengths of native and denatured Anacy_4718g3 assembled with PEB, PCB, or PΦB chromophores for the 15Z configuration of PCB and PΦB (FIG. 15C). This correlation implies that spectral tuning of all three chromophores by Anacy_4718g3 is comparable, despite the lack of a conjugated D-ring in PEB. Therefore, these results establish the D-ring as the site of primary photochemistry and the A-, B-, and/or C-rings as the site of the pronounced red shift induced by Anacy_4718g3. Spectral tuning and photoconversion of the far-red-absorbing photostate thus occur at different locations within the bilin chromophore.

Example 4. Fluorescence Properties of Far-Red/Orange CBCRs

The peak absorption observed for far-red CBCRs is well into the far-red/near-IR window of maximum penetrance in animal tissues.[31] The only biliproteins known to absorb at longer wavelengths are BV-containing bacteriophytochromes in the 15E P_(fr) state.[79] Unfortunately, known phytochromes exhibit little to no fluorescence from the P_(fr) state, with extremely short-lived excited states.[85-87] The 15Z far-red states of these newly characterized CBCRs might therefore exhibit higher near-IR fluorescence, because the bilin chromophore is in the same 15Z configuration found in phytochromes engineered for higher fluorescence quantum yield.[21-24, 88] The far-red/orange CBCRs were the focus of subsequent studies because the two photostates in such proteins have greater spectral separation.

All three far-red/orange CBCRs were characterized by fluorescence spectroscopy. These measurements were complicated by the presence of porphyrin (FIG. 10A, asterisk), and fluorescence from the far-red state of Anacy_4718g3 could not be detected against this background (FIG. 14A). Far-red and near-infrared fluorescence could be detected for both Anacy_2551g3 and Cyan7822_4053g2 (FIG. 14B-C). Interestingly, the excitation spectrum of Anacy_2551g3 contained multiple peaks (FIG. 16A) that were ascribed to heterogeneity of the far-red state. Fluorescence emission of Anacy_2551g3 extended well into the near-IR (FIG. 16A-B). The observed Stokes shift for the far-red/near-IR state was small, with fluorescence emission peaking at approximately 740 nm for both Anacy_2551g3 and Cyan7822_4053g2. This Stokes shift followed the general trend seen for other CBCRs (FIG. 14D). The fluorescence quantum yield for Anacy_2551g3 was estimated at 1.2% using the ratio method,[22] plotting integrated emission versus absorbance for a dilution series with Alexa 750 as standard (FIG. 16C). These results establish far-red CBCRs as fluorescent, with some examples exhibiting modest near-IR fluorescence detectable at very long wavelengths.

The work described herein reveals two conserved clusters of far-red CBCRs. Three such proteins belong to the first cluster and exhibited similar far-red/orange photocycles. Two of these proteins also exhibited detectable near-infrared fluorescence from the far-red-absorbing state. Two other proteins belong to the second cluster and exhibited similar far-red/red photocycles. Denaturation analysis demonstrated that far-red sensing at 728-740 nm utilized a covalent 15Z PCB chromophore similar to that found in other CBCRs and in cyanobacterial and algal phytochromes.[42, 46, 47, 51, 54, 81, 82, 89] Remarkably, this indicates that the same chromophore precursor provides CBCRs with the ability to detect light ranging from 330 to 740 nm through diverse tuning mechanisms. CBCR tuning mechanisms characterized to date provide strategies for blue shifting peak absorption relative to a protonated, cationic bilin 7C system.[17, 43, 47, 71, 73] The extreme red shift reported here therefore implies the existence of a previously unrecognized tuning mechanism.

Although there are parallels between far-red CBCRs and the far-red-absorbing P_(fr) states of phytochromes, there are also striking differences. It is therefore unclear whether the same tuning mechanisms are responsible for far-red peak absorption in phytochromes and CBCRs. Far-red CBCRs absorb far-red light in the 15Z chromophore configuration rather than the 15E configuration of the phytochrome P_(fr) state.[90, 91] Moreover, most phytochromes require both the bilin-binding GAF domain and the adjacent PHY domain for P_(fr) formation,[22, 32, 92, 93] whereas far-red CBCRs lack PHY domains altogether (FIG. 17). Previous studies indicate that different phytochromes generate the P_(fr) state in different ways,[65] and no model explaining far-red absorption of PCB or PΦB in the phytochrome P_(fr) state is generally accepted at present.[94] A variety of models for the 15Z far-red-absorbing state of these newly described CBCRs should therefore be considered.

Denaturation analysis confirms that far-red CBCRs employ 15,16-photoisomerization as in other CBCRs,[37, 39, 58, 59] establishing D-ring rotation as the site of photochemistry. However, the photochemically inactive PEB adduct of Anacy4718g3 exhibits a red-shifted 15Z state comparable to those seen for the other two bilin adducts (FIG. 15C). The D-ring in PEB is not conjugated with the rest of the chromophore (FIG. 1), so the red shift of the PEB adduct must be due to protein-dependent perturbations of the conjugated ABC-ring system. By contrast, the red shift of the PΦB adduct of Anacy4718g3 relative to that of its PCB adduct is comparable to those seen in other CBCRs (FIG. 15A). Therefore, the tuning mechanism generating far-red absorption in Anacy_4718g3 and related proteins is independent of conjugation between the C- and D-ring.

Far-red CBCRs could use a tuning mechanism that does not occur in phytochromes. For example, the far-red state could arise due to formation of the lactim tautomer at the A-ring, consistent with the known red shift of O-alkylated bilins and inconsistent with the known protonation state of P_(fr) phytochrome.[90, 95, 96] Anionic bilin 7C systems also exhibit substantial red shifts[97, 98] but are again incompatible with the known P_(fr) protonation state. Without wishing to be bound by any particularly theory, it is believed that an anionic bilin could be stabilized by a bound metal ion, but the presence of such a cation in far-red CBCRs has not yet been directly observed. The presence of multiple conserved Trp residues in far-red/orange CBCRs (FIG. 3) is consistent with the role of Trp residues in red-shifting phycobiliprotein chromophores,[99] although the red shift observed in phycobiliproteins is much smaller than that observed in this work. The presence of Trp residues proximal to the chromophore also raises the possibility of a previously unknown charge-transfer process generating a labile species not observed in the denaturation assay, a situation somewhat analogous to charge-transfer processes in the blue light receptor cryptochrome.[100, 101] Any of these mechanisms could explain the anomalously low extinction coefficients observed for far-red states described in this study (FIG. 15B), because the resulting chromophore structures would chemically differ from the protonated PCB and phycoviolobilin chromophores that establish the observed trend. Consistent with this point, the other exception for the general correlation between extinction coefficient and peak wavelength observed in 15Z PCB adducts is green-absorbing states such as that of RcaE, which is known to have a deprotonated bilin ring system.[17] Elucidating the basis for far-red sensing in these proteins will thus require further studies.

The far-red/orange and far-red/red CBCR photocycles described here imply that members of the far-red/orange cluster have a distinct mechanism for spectral tuning of the orange-absorbing photoproduct. The orange-absorbing photoproducts observed in Anacy_4718g3, Anacy_2551g3, and Cyan7822_4053g2 are very similar, exhibiting a slight blue shift relative to denatured 15E PCB adducts and a characteristic lineshape (FIG. 6). Moreover, no red shift is observed in the Anacy_4718g3-PΦB photoproduct relative to the Anacy_4718g3-PCB photoproduct (Table 2). The absence of such a PΦB blue shift has also been observed in the teal-DXCF CBCR lineage, in which the photoproduct D-ring is trapped in a twisted geometry reducing conjugation to yield a blue-shifted chromophore with a similar lineshape.[71]

It is thus possible that a similar trapped-twist mechanism acts to tune the orange-absorbing photoproduct. To examine this hypothesis, 15E photoproduct blue shift was plotted versus 15Z peak wavelength for a range of trapped-twist CBCRs, for a range of CBCRs lacking residues required for trapped-twist photoproducts and hence having ‘relaxed’ photoproducts, and for denatured samples.[51, 53, 71] CBCRs with trapped-twist photoproducts exhibited a linear relationship between these spectral parameters (FIG. 15D), and far-red/orange CBCRs followed this correlation. Relaxed CBCRs were more similar to denatured samples, and both far-red/red CBCRs examined in this study followed the relaxed trend (FIG. 15D). This analysis implicates some type of trapped-twist tuning in the orange-absorbing photoproducts of far-red CBCRs. Interestingly, comparison of the Asp-motifs of far-red/red CBCRs to those of far-red/orange CBCRs reveals that far-red/red CBCRs lack one of the Trp residues found in far-red/orange CBCRs (FIG. 3). This Trp residue may thus constrain chromophore motions during photoconversion. Aromatic residues are similarly implicated in spectral tuning in other CBCR lineages,[53, 71, 102] and recent work also implicates Trp residues in spectral tuning of phycobiliproteins.[99] It is thus possible that a similar effect provides spectral tuning in far-red/orange CBCRs.

The work described herein also implicates additional unknown far-red to near-IR photobiology in cyanobacteria. The newly recognized far-red CBCRs do not correlate with known far-red photobiological responses. For example, the filamentous cyanobacterium Leptolyngbya sp. JSC-1 is known to exhibit far-red light-induced photoacclimation (FaRLiP), but this organism lacks a far-red CBCR (Tables 1 and 3) and FaRLiP is controlled by a different photosensor.[93, 103-105] The more recently described low-light-induced photoacclimation (LoLiP) response also does not correlate with FR CBCRs, as shown by the presence of a far-red CBCR (Table 1) but absence of LoLiP in Leptolyngbya sp. PCC 7104.[104] Far-red CBCRs are present in both unicellular and filamentous cyanobacteria and are associated with multiple histidine kinase lineages and domain architectures (FIG. 2 & Tables 1 and 3). By contrast, the two known classes of CCA regulator are associated with coherent phylogenetic clusters in both the CBCR and histidine kinase trees (FIG. 2), and the RcaE cluster correlates well with the presence of type III CCA in the host organisms (Table 5). Far-red CBCRs are therefore likely to be associated with multiple physiological responses.

TABLE 5 CCA properties for cyanobacteria hosting RcaE-type CBCRs. cyanobacterium green/red CBCR CCA type Staniera cyanosphaera Sta7437_2963 III PCC 7437 Synechococcus sp. PCC 7336 WP_017324438 III (Syn7336_0672) Xenococcus sp. PCC 7305 X7305_GR III (Xen7305DRAFT_00010580) Leptolyngbya sp. JSC-1 JSC1_30280 III (CYJSC1_DRAFT_30280) Tolypothrix sp. PCC 7601² RcaE (fdiDRAFT24240) III Synechococcus sp. PCC 7335 S7335_5042g1 III (S7335_5042)

In Table 5, strains hosting the RcaE cluster identified in FIG. 2 are reported where information about CCA is available. Both the protein tag used in FIG. 2 and the DOE-IMG locus tag are listed when they are different. Information about strains in the Pasteur Culture Collection (PCC) is taken from the PCC catalog, and information about Leptolyngbya sp. JSC-1 is taken from Gan & Bryant [104]. Tolypothrix sp. PCC 7601 in Table 5 is also known as Fremyella diplosiphon.

The far-red CBCRs characterized here can be used as fluorescent reporters, optical contrast agents, or synthetic biology reagents responding to far-red or near-infrared light. Far-red CBCRs exhibit a unique combination of far-red peak absorption and detectable near-infrared fluorescence not found in other phytochromes or CBCRs. These proteins thus hold great promise as fluorescent reporters and optical contrast agents in systems for which the far-red/near-IR window is critical for optimal performance. It should be possible to engineer far-red CBCRs with improved affinity for BV and/or higher fluorescence QY.[21, 23-25] It should also be possible to couple far-red CBCRs to alternative outputs for modulating various aspects of eukaryotic biology, as has been done for bacteriophytochromes.[29, 30] Far-red CBCRs thus extend a series of studies[40, 42, 45-49, 51-54, 106] establishing CBCRs as having the broadest light sensing range of known photoreceptor families. As modular light sensors ranging from ca. 330 nm to 750 nm, CBCRs span the full range of the electromagnetic spectrum amenable to oxygenic photosynthesis by cyanobacteria.

Example 5. Far-Red CBCR-Annexin Fusion Construct for In Vivo Detection of Apoptosis

Apoptotic processes are studied in vivo by fusing the Anacy_2551g3 CBCR domain to annexin V, a well-established apoptotic reporter, using recombinant DNA technology. Annexin V binds to phosphatidylserine (PS), which is normally not present on the extracellular leaflet of the mammalian plasma membrane. Induction of apoptosis results in the loss of asymmetric lipid distribution and the appearance of extracellular PS. Fluorescent conjugates of annexin V are thus widely used as apoptotic markers in fluorescence microscopy and cell sorting, often being generated by derivatization with reagents such as fluorescein isothiocyanate (FITC). Application of FITC-annexin V to whole animals is more difficult, because fluorescein fluorescence is not in the window of optimal transmission for mammalian cells. The use of a far-red CBCR fused to annexin V alleviates this difficulty and also permits the use of annexin V markers in the presence of other fluorescent reagents that normally overlap with FITC fluorescence, such as green fluorescent protein (GFP).

DNA encoding Annexin V is procured from a commercially available pJM31 plasmid (Addgene) or by commercial gene synthesis. The annexin V sequence is then fused to the Anacy_2551g3 CBCR domain in either orientation (i.e., CBCR-annexin or annexin-CBCR) and the resulting fusion construct is purified after heterologous expression in E. coli. The fusion construct is then used as a reagent for imaging in tissue culture or whole animals. Examples of annexin-Anacy_2551g3 images of HEK cells are shown in FIG. 20 and discussed further in Example 6 below.

Example 6. Annexin-Anacy_2551g3 Fluorescence Imaging in HEK 293 Cells

In this example, an annexin-Anacy_2551g3 protein fusion construct was expressed and purified, and then the concentrated purified protein (25X) was used for imaging in HEK 293 cells.

HEK 293 cells were treated with camptothecin at a concentration of 5 μM for 4 hours to induce apoptosis. Then the cells were treated with 10 μL, 20 μL, or 30 μL of 25× stock protein fusion construct solution for 15 minutes. Cells were subsequently imaged with a 40× air objective, wide-field microscope (i.e. Zeiss observer) and excited with Xcite LED RDX (660-675 nm, filter set cy5.5). As a control, FITC-annexin was added for co-staining (i.e., added at 15 minutes post annexin-Anacy_2551g3 incubation). Representative images are shown in FIG. 20.

A GFP filter was usted to identify cells that were FITC-positive, and then imaging was performed using all three channels. The images in the top row (i.e. “cell1”) are depicted using pseudo-color, while the images in the bottom row (i.e. “cell2”) are depicted in original format. As a result of the apoptotic cells becoming detached, they were not entirely within the imaging focal plane, also demonstrating that the fluorescent signal was mostly localized at the cell surface.

Example 7. In Vivo Imaging in Phantom Mice Using Purified Anacy_2551g3

To demonstrate the utility of these new CBCRs for in vivo imaging techniques, purified Anacy_2551g3 was used in two systems that mimic real-world imaging modalities: fluorescence imaging in a widely used phantom mouse model (FIGS. 18A-18C) and photoacoustic (PA) imaging of silicone tubes mounted under chicken meat to mimic the scattering properties of animal tissue (FIGS. 19A-19C).

For the phantom mouse experiment, a commercial IVIS system (Perkin-Elmer) and its associated phantom mouse were used to demonstrate the potential of these proteins for fluorescence imaging in whole animals with off-the-shelf components. Using wavelengths of 710 nm for excitation and 760 nm for emission, Anacy_2551g3 could be detected at a depth of 18.1 mm in the phantom animal (FIG. 18B). Surprisingly, fluorescence could also be detected at a depth of 7 mm using wavelengths of 745 nm for excitation and 800 nm for emission (FIG. 18C), demonstrating the ability of near-infrared (NIR) CBCRs to function as reagents for in vivo fluorescence imaging.

For the PA experiment, data was obtained using a PA system with purified Anacy_2551g3 mounted in transparent silicone tubes that were either immersed in water or placed under 8 mm of highly scattering media (i.e., chicken tissue). For PA imaging and photoconversion of the far-red “ON” dark-adapted state to the orange-absorbing “OFF” photoproduct state, 728 nm laser illumination was used. 588 nm laser light was used to regenerate the ON state, matching photoproduct absorption. PA signals could readily be observed over multiple switching cycles (FIG. 19A) and even under 8 mm of tissue (FIG. 19B). The signal ratio was defined as the ratio of the mean protein signal to the mean blood signal. In the ON image, Anacy_2551g3 had a comparable signal ratio to that of blood. However, the signal ratio was above unity at both 0 mm (˜11 fold) and 8 mm (˜2 fold) depth in differential images (FIG. 19C), demonstrating the ability of NIR CBCRs to function as contrast agents for in vivo PA imaging.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. Where a conflict exists between the instant application and a reference provided herein, the instant application shall dominate.

VI. References

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VII. Exemplary Embodiments

Exemplary embodiments provided in accordance with the presently disclosed subject matter include, but are not limited to, the claims and the following embodiments:

-   -   1. A protein fusion construct comprising a far-red         cyanobacteriochrome (CBCR) domain linked to a heterologous         domain, wherein the far-red CBCR domain comprises a CBCR         polypeptide and a tetrapyrrole chromophore.     -   2. The protein fusion construct of embodiment 1, wherein the         CBCR polypeptide comprises a GAF domain having an acidic motif         comprising:     -   a conserved tryptophan residue; followed by     -   two acidic amino acid residues, wherein at least one of the         amino acid residues is an acidic amino acid residue; followed by     -   a conserved glutamic acid residue; followed by     -   a further amino acid residue; followed by     -   an aromatic amino acid residue.     -   3. The protein fusion construct of embodiment 2, wherein the         acidic motif comprises an amino acid sequence set forth in SEQ         ID NO: 1:

(1) W-X9-X6-E-X1-X5

-   -   wherein:     -   W is a tryptophan residue;     -   E is a glutamic acid residue;     -   X1 is an independently selected amino acid residue;     -   X5 is independently selected from the group consisting of a         phenylalanine residue, a tyrosine residue, and a tryptophan         residue;     -   X6 is independently selected from the group consisting of an         aspartic acid residue and an asparagine residue; and     -   X9 is independently selected from the group consisting of an         aspartic acid residue and a glutamine residue.     -   4. The protein fusion construct of embodiment 1, wherein the         CBCR polypeptide comprises a GAF domain having an acidic motif         comprising:     -   a hydrophobic residue independently selected from the group         consisting of an alanine residue, a cysteine residue, a valine         residue, a threonine residue, a leucine residue, an isoleucine         residue, a methionine residue, a phenylalanine residue, a         tyrosine residue, and a tryptophan residue; followed by     -   a further amino acid residue; followed by     -   a conserved aspartic acid residue; followed by     -   a conserved glutamic acid residue; followed by     -   a further amino acid residue; followed by     -   a hydrophobic residue independently selected from the group         consisting of an alanine residue, a cysteine residue, a valine         residue, a threonine residue, a leucine residue, an isoleucine         residue, a methionine residue, a phenylalanine residue, a         tyrosine residue, and a tryptophan residue; followed by     -   a proline residue.     -   5. The protein fusion construct of embodiment 4, wherein the         acidic motif comprises an amino acid sequence set forth in SEQ         ID NO: 2:

(2) X2-X1-D-E-X1-X2-P

-   -   wherein:     -   each X2 is independently selected from the group consisting of         an alanine residue, a cysteine residue, a valine residue, a         threonine residue, a leucine residue, an isoleucine residue, a         methionine residue, a phenylalanine residue, a tyrosine residue,         and a tryptophan residue;     -   X1 is an independently selected amino acid residue;     -   D is an aspartic acid residue;     -   E is a glutamic acid residue; and     -   P is a proline residue.     -   6. The protein fusion construct of any one of embodiments 1-5,         wherein the CBCR polypeptide comprises an amino acid sequence as         set forth in SEQ ID NO: 3:

(3) X9-R-X1-X3-X4-F-X1-X3-(X1)2-X6-G-(X1)3-X4-X2-E-E- X1-V-(X1)3-X2-(X1)2-X2-(X1)4-W-X8-X6-E-X1-X5-X1- X7-X9-(X2)2-X8-X2-Y-X1-Q-G-X1-P-R-I-V-X1-X6-V-X2- X10-X1-D-X1-X5-X2-X1-C-L-X1-E-X5-(X1)5-X4-X1-S-K- X4-V-A-P-I-X2;

-   -   wherein each A is an alanine residue, each C is a cysteine         residue, each D is an aspartic acid residue, each E is a         glutamic acid residue, each F is a phenylalanine residue, each G         is a glycine residue, each I is an isoleucine residue, each K is         a lysine residue, each L is a leucine residue, each P is a         proline residue, each Q is a glutamine residue, each R is an         arginine residue, each S is a serine residue, each V is a valine         residue, each W is a tryptophan residue, and each Y is a         tyrosine residue;     -   and wherein:     -   each X1 is an independently selected amino acid residue;     -   each X2 is independently selected from the group consisting of         an alanine residue, a cysteine residue, a valine residue, a         threonine residue, a leucine residue, an isoleucine residue, a         methionine residue, a phenylalanine residue, a tyrosine residue,         and a tryptophan residue;     -   each X3 is independently selected from the group consisting of a         valine residue, a leucine residue, an isoleucine residue, and a         methionine residue;     -   each X4 is independently selected from the group consisting of a         valine residue, an isoleucine residue, and a threonine residue;     -   each X5 is independently selected from the group consisting of a         phenylalanine residue, a tyrosine residue, and a tryptophan         residue;     -   each X6 is independently selected from the group consisting of         an aspartic acid residue and an asparagine residue;     -   each X7 is independently selected from the group consisting of a         glutamic acid residue and a glutamine residue;     -   each X8 is independently selected from the group consisting of         an aspartic acid residue, an asparagine residue, a glutamic acid         residue, and a glutamine residue;     -   each X9 is independently selected from the group consisting of         an aspartic acid residue and a glutamate residue; and     -   X10 is absent or X10 is one or more independently selected amino         acid residues.     -   7. The protein fusion construct of embodiment 6, wherein X10 is         absent.     -   8. The protein fusion construct of any one of embodiments 1-7,         wherein the CBCR polypeptide comprises an amino acid sequence         set forth in:     -   SEQ ID NO: 4 (Sta7437_1656),     -   SEQ ID NO: 5 (Cyan7822_4053g2),     -   SEQ ID NO: 6 (Anacy_2551g3), or     -   SEQ ID NO: 7 (Anacy_4718g3).     -   9. The protein fusion construct of any one of embodiments 1-7,         wherein the CBCR polypeptide comprises an amino acid sequence         set forth in:     -   SEQ ID NO: 4 (Sta7437_1656),     -   SEQ ID NO: 5 (Cy7822_4053g2);     -   SEQ ID NO: 6 (Anacy_2551g3);     -   SEQ ID NO: 7 (Anacy_4718g3);     -   SEQ ID NO: 8 (N7104D_1016g3);     -   SEQ ID NO: 9 (L6406D_1154g2);     -   SEQ ID NO: 10 (c56D2_02270g2);     -   SEQ ID NO: 11 (c407D_01196g2);     -   SEQ ID NO: 12 (fdiDRAFT29700);     -   SEQ ID NO: 13 (WP009627289g3);     -   SEQ ID NO: 14 (Os7112_5903g3);     -   SEQ ID NO: 15 (C6303_3693g3);     -   SEQ ID NO: 16 (WP006632756g3);     -   SEQ ID NO: 17 (Cy7425_1390g3);     -   SEQ ID NO: 18 (WP017296986g2);     -   SEQ ID NO: 19 (WP_033374293);     -   SEQ ID NO: 20 (WP028089844g3);     -   SEQ ID NO: 21 (WP008316973g2);     -   SEQ ID NO: 22 (Ga0039499_10213);     -   SEQ ID NO: 23 (310F_3509);     -   SEQ ID NO: 24 (WP_016871037);     -   SEQ ID NO: 25 (WP_016878855);     -   SEQ ID NO: 26 (WP_026722600);     -   SEQ ID NO: 27 (WP_017309337); or     -   SEQ ID NO: 28 (WP_016873240).     -   10. The protein fusion construct of any one of embodiments 1-9,         wherein the tetrapyrrole chromophore is a bilin.     -   11. The protein fusion construct of embodiment 10, wherein the         bilin is selected from the group consisting of phycocyanobilin         (PCB), phytochromobilin (PΦB), phycoerythrobilin (PEB), and         biliverdin (BV).     -   12. The protein fusion construct of any one of embodiments 1-11,         wherein the C-terminus of the CBCR polypeptide is linked to the         heterologous domain.     -   13. The protein fusion construct of any one of embodiments 1-11,         wherein the N-terminus of the CBCR polypeptide is linked to the         heterologous domain.     -   14. The protein fusion construct of any one of embodiments 1-13,         wherein the heterologous domain comprises a heterologous         oligopeptide or a heterologous polypeptide.     -   15. The protein fusion construct of any one of embodiments 1-13,         wherein the heterologous domain comprises a heterologous         polypeptide selected from the group consisting of a signaling         polypeptide, a structural polypeptide, a transport polypeptide,         a targeting peptide, a hormone polypeptide, and a regulatory         peptide.     -   16. The protein fusion construct of embodiment 15, wherein the         signaling polypeptide is selected from the group consisting of a         kinase polypeptide, a phosphatase polypeptide, a         phosphodiesterase polypeptide, a nucleotide cyclase polypeptide,         a protease, a phopholipase, a G-protein polypeptide, and a         channel protein polypeptide.     -   17. The protein fusion construct of embodiment 15, wherein the         structural polypeptide is selected from the group consisting of         an actin polypeptide, a tubulin polypeptide, a myosin         polypeptide, and a collagen polypeptide.     -   18. The protein fusion construct of embodiment 15, wherein the         transport polypeptide is selected from the group consisting of         an annexin polypeptide and a clathrin polypeptide.     -   19. The protein fusion construct of embodiment 15, wherein the         targeting polypeptide is selected from the group consisting of         an antibody, an antibody fragment, and an aptamer.     -   20. The protein fusion construct of any one of embodiments 1-13,         wherein the heterologous domain comprises a streptavidin         polypeptide.     -   21. A method for detecting a cellular component, the method         comprising:     -   providing a protein fusion construct in a sample, the fusion         construct comprising a far-red CBCR domain and a heterologous         domain specifically detecting a cellular component;     -   exposing the protein fusion construct to far-red light or         near-IR light, wherein the exposing causes fluorescence of the         far-red CBCR domain; and     -   detecting the fluorescence of the far-red CBCR domain,     -   thereby detecting the cellular component.     -   22. The method of embodiment 21, wherein the sample is a cell or         tissue and wherein providing the protein fusion construct         comprises expressing the protein fusion construct in the cell or         tissue.     -   23. A method for imaging a biological structure in a subject,         the method comprising:     -   providing a protein fusion construct in or near the biological         structure, the fusion construct comprising a far-red CBCR domain         and a heterologous domain;     -   exposing the protein fusion construct to far-red light or         near-IR light, wherein the exposing causes absorbance by, or         release of an acoustic signal or fluorescence from, the far-red         CBCR domain;     -   detecting the absorbance, acoustic signal, or fluorescence of         the far-red CBCR domain; and     -   constructing an image of the biological structure;     -   thereby imaging the biological structure.     -   24. The method of embodiment 23, wherein the biological         structure is selected from the group consisting of a tissue, an         organ, or a tumor.     -   25. A method for modulating a cellular process, the method         comprising:     -   expressing a protein fusion construct in a cell, the fusion         construct comprising a far-red CBCR domain and a heterologous         signaling domain;     -   exposing the protein fusion construct to far-red light or         near-IR light;     -   wherein the exposing increases or decreases the activity of the         heterologous signaling domain, thereby modulating the cellular         process.     -   26. The method of embodiment 25, wherein the heterologous         signaling domain is selected from the group consisting of a         kinase polypeptide, a phosphatase polypeptide, a         phosphodiesterase polypeptide, a nucleotide cyclase polypeptide,         a protease, a phopholipase, a G-protein polypeptide, and a         channel protein polypeptide.     -   27. The method of any one of embodiments 21-26, wherein the         far-red cyanobacteriochrome domain comprises a CBCR polypeptide         and a tetrapyrrole chromophore.     -   28. The method of embodiment 27, wherein the CBCR polypeptide         comprises an amino acid sequence set forth in any one of SEQ ID         NOS: 1-28.     -   29. The method of embodiment 27 or 28, wherein the tetrapyrrole         chromophore is selected from the group consisting of         phycocyanobilin (PCB), phytochromobilin (PΦB), phycoerythrobilin         (PEB), and biliverdin (BV).     -   30. An isolated nucleic acid comprising a polynucleotide         sequence encoding the protein fusion construct of any one of         embodiments 1-20.     -   31. An expression cassette comprising the nucleic acid of         embodiment 30 operably linked to a promoter.     -   32. The expression cassette of embodiment 31, further comprising         at least one nucleic acid encoding an enzyme for chromophore         synthesis.     -   33. A vector comprising the nucleic acid of embodiment 30.     -   34. A host cell comprising the nucleic acid of embodiment 30,         the expression cassette of embodiment 31, or the vector of         embodiment 33.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

INFORMAL SEQUENCE LISTING

The following Informal Sequence Listing provides exemplary sequences of full-length proteins containing far-red CBCRs. Bold and underlined regions correspond to the core CBCR regions aligned in FIG. 3.

Anacy_4718 (Bold and underlined region = SEQ ID NO: 7) (SEQ ID NO: 29) MFSRSVILTPSELKSAIIRNPLIVKPETTVIDAIAMLAAGIGQMGGVGAI SNTKIIDGQLDELHLETRPSCVLVMEDGKLLGIFTERDVVRLISQQHSLE NLVIQDVMTYPVVTLYESAFSDLFSTINLLQQYHIRHIPILNEQDCVVGL VTDESLRQISNPIYPLRSRLVSAAMTNEVICAALDSSIRTIVQLMAKNCI SCVIIVQKRGSQAQPLQIPVGIITEQDIVKFQVLGLNLETSQAETVMSAP IFSVKPNDSLEMVQQIMEQQLIRKLAVTNEEGNLLGIVTQNSLLQTLNPL ELYKLAEVLEEKVLRLEAEKILLLETRTVELEKELADQNIALQTKTEQEK LVAIIATQIRSSLNLQTILDTTVEQIRQLLNCDRVTIWQLEANGKLITVA ESTGCTLSLLGQQSQDQCISQQLVEIYQQGKIRIVPDIYTTEMSDCHRNL LISLDIRAKILMPLMCGDELWGFLNVTESQHPRQWQDSEIELLKLLTVQL AIALQQATTHQKLQEELRERQRAESTLQKLVTGTAAVTGDDFFPALVQHI AEALDVSYAIVTELVGDQLHTLGFWANGSLQPSVSYYAAHTPCKYALRDG QFYCKSGIQEAFANDFDLVMMRADSYLGIALKDDLGNAIGNLCILDVQPL HNSQLKEARDILQVFAARAAAELQRKIAKDALISLNHNLELRIEQRTTKL QAREAQLRDLFDNATDLIQSISLNGRILFVNKSWKEALGYDDTDLEKLSI FQVIHPDELVHCQTVMASLASGNPSMSMETRFLTKDGREIIVEGNVNCQF AKGKPIATRGVFRDITQRKQAELALEEAQQFLYTVLDTFPLFIFWKNRES VYLGCNQNFAISGGFASPAEVIGKTDDDFPWRNGEADIYRADDRQVIESG IAKLGIIETQQQTNGSTIWLETNKLPLRNLKGEVIGILGTYQDITERKQA ENALQNSELRFRRMFDSSVVGMIFADFQGRILDTNDRFLQMLGYTRDDFN AGAINWLAITPSEYIPTDFAAIDHLMKYGEIDPWEKAYYRQDGSRIPVLI GAAILPESKDQTICVVVDISEQKAALRERQEAELSLQQEAMYKQLLLTLS QAIRESLEIEVILNTSVNEARSLLVVDRVAVYRFQPDWSGEFITEAVVPG WVKLAAESDVKKVWQDTYLQETQGGRFRNYETLIVRDIYQAGLQPCHIEL LEQFQARAYVITPIFVGESLWGLLGMYQNDQPYWWTTGEIELLQQIASQL AIAIYQANLYQQVQAELIIRQKAELAISHQLQQQRTLGKIVQKIRDSLDI KDILATVTQEIKNSLNC DRVIVFRLFADGESQIVEEAVSGELVSLKNRHW DNEVWSQEILDYYWQGQPRIVTDVMEDIWTDCLVEYSIEGQVQSKIVAPI LQEAQDGEKNRWVASGENNKLWGVLVVH ACSEKRIWKDCEAQLLQQIANQ LAIAIQQANLFEKLQQELTERQKTEIKLTHSNQQLAISNEELARATRLKD EFLANMSHELRTPLNAILGMTEALQEQVFGGINERQLKALKTVENSGNHL LELINDILDLAKIEAGQINLNCTSISVSHLCQSSLAFIKQQALKKRIKLN IKLPQNLPDLFVDERRIRQVLINLLNNGVKFTPQGGSITLEVTQFHPDME NADFFPQGFLRITVIDTGIGISPENINRLFHPFIQIDSSLSRQYNGTGLG LALVKQIVELHGGQVGLTSELGVGSCFMIDLPCSPLLSEITTDDQSASTS ELDFLTAEEAESQAPLILLAEDNEANIITFSSYLEAKGYQIILARDGHEA VNLAKTHQPNLILMDIQMPGMDGLEAMTQIRLDPKLINIPIIALTALAMT GDRERCLEAGANDYLTKPVMLKQLATTIQQLLNKDG Anacy_2551 (Bold and underlined region = SEQ ID NO: 6) (SEQ ID NO: 30) MFVPAKAHTQAELRLAIVRDPLLVTPETTVVEAIAQMSGVRSVCSVSQNT DSKQESLLAEARSSCVVVVENNQPIGIFTERDVVRLSAQRRKLNNLAIRD VMAYPLVTLYESEFTDLFFAINLLQHHKIRHLPVIDEQNQLVGLLTHESL RQKSRPVDLLRLRLVSEVMTTKVICAAPHISILMIARLMAENRISSVVIV QTQASLIIPIGIVTERDIVQFQALDLNFETCLAEAVMSTPIFCVNADESL WNVQQIMEQRLIQRLAVTGTNGELLGIVTQSSILQVLNPLELYKLTELLE KKVSQLEAEKIELLENRTVELEEEVGERTIALRKKIVREQLITKIAAQIR SSLNLQDILNTTVAEMRSLLQCDRVIIYQFRPDFSGTVIAESIVANGVSI LHDEPQDPCITPEWLEPYRQGQIRVINDIHSESMSDCYQDMLIELDIRAK LMVPIVIAEQLWGLILTSYRDQSHNWELEEIELVRQLSIQLSVAIQQAQT HQQLYQLNQELENQIQERTKALQASEAKYRNLVEAATHVTWLCNTKGELI YLSPQFQELFGWEVEKFYGQSFISLIHPDDRPYMISTSEELGKSDKNLVS AEFRHLHQNGSYIWVESKASNLKDASGVIIGCQGVLLDISDRKQAEKIIK QQAEREHLLYQTTQRIRQSLDLATIFNTATQEIRQFMNADRVVIFQLDPV SNFNDSKFVSESVVEGFTSALATKINNKCFGEQYAAHYQQGRIQVVDDLD NAGLTDCHRDVLAQFQVRANLVVPLLQGENLWGLLCIHQCSVPRHWQEFE VELVQQIAHQLAIAIQQSILYEQVQSELIIRKQAEDAISLQLQRQKIIQD ITQQIRSTLNVNHILATVTQQVKELMQV ERVIIFRLFPNGRSQIVEEVVS SEYAALKNYHWEDEKWSQEILDCYWQGKPRIVPDVINDIWTSCLVEYTTQ GNIQSKIVAPILQELGENETGRWVSSEHKQKLWGVLVVH ACSTKRVWEED EAQLLQQIANQLAIAIQQAALFEQLQLSLVQEKEVSKMRSRFITMASHEF RTPLAIIASSTGILQKFRERLSAEKQQEHLGTIQKTIKHIIQLLDDVLMI NRTEAEKMEFKPEASDIIAFCHQITQQIEATSNKHVIEFSFTASKPILDN SFIVQLDKKILQQILANILTNAIKYSPQTSLIKFDLTIEDDKLIFKIKDS GIGIPEEYKINLFAPFHRASNVGTISGTGLGLSIVKKCVDLHKGEISFDS KLGQGTTFTIIIPYSRIQESGVRSQE 310F_3509 (Bold and underlined region = SEQ ID NO: 23) (SEQ ID NO: 31) MLIPVNAISATELKSAITYNPLLATADTTVREAVVQMSGISANTLWLNVP AVCYIPQTANNYLEHLQIQACCSCVLIVENNRPVGIFRQEDVVKISTQKP NLEDLALRDVMTHPVITLEESKFTDLFSALNLLQHHRIRHLPLVDEENQL VGLLTYESLRQILRPVDLLRLRLVHEVMTTNVLCAPANVSILEIARLMAE NQVSSVMIVETQASLTIPLGMVTEHDIVQLKALSVNFDTCLAQTVMSTPV FCVTVDESLWNVQQIMEQRFIQLLAVTGSKGELLGIVTHSSILEALNPWE LYKLTAVLQEKVLQLETEKIQLLENRTLELEKQIEERTIKLRRKAEQEEL INQIATQIHSSLDLQEILNNTVVGVRSLLNCDRVIVYQFSGDFRGQVIAE AIITGESVLNQEVHDPCISPEWLELYRQGQIRVINDINTESITQCHQQML KDLDIRGKLLSPLIVENQLWGLMLASYRDIPHNWELEEIELVQQISLRVA IAIQQANIYQQTQIEIHQRQQAEELIKQQLAELKIWKNRYELASTVSGQI MYEYNLLNDAPVWPANMEEILGYSYSECPRNLAEFMDIVHPEDRDRLYSL IQKKLAHKSPLSTEYRLRRKDGNYIWVEDRNQVVLDDQGEIVVVIGAIVD ITVRKNSEEKLSKLFQKSEKLQERLSLVLKGSNDAWWDWDLLEDTIYYSA RWFSLLGYKHEELYLKSESFWQNFMHPEDIDPIRGNFNQALDDKNIEFIE SKFRLRHKQEYYIFINCRSYILRDETGKAVRVSGANTDITQLVQKEEELQ ATLNQLSQFNQKLETRVQKRTVQLQNLSSRLELAIKAAKIAIWEWDLDNN HTIWDKKMYELYGVKPSEYKDGMEILQTVLHPEDAVRVNEILQHKLKDGE EFEMDFRIVLPDGKIRVLQSYGIIKRDSQGKAERVIGVNKDITEWKQTEQ KIKQQAEREYLLWETTQRIRQSLDLHTIFNTAAAEIRQLMNADRVGIFKF DPDSNFNYGEFVSESVVPGFISALEMKVHDQCFGERFSSDYAAGRMQIVD DIDNAGLADCHRDILAQFQVRANLVVPLIQGRILWGLLCIHQCSQPRHWE DFEIELVQQIANQLAIAIQQSMLYEQVQSELIIRKQAEVEIYLQLQRQRA IQDITQEIRSSLNLNHILTTITAKVQELTKA ERVIVFRLFPDGKSQIVEE AVANGYMTFKDSYWEDEKWSQDILEYYWQGKPRIVLDVMDDIWTDCLKAY SRQGNIRSKIVAPILQDLVENENGRWVNHPHNKLWGVLVVH ACGEKRIWE ESEAELLQQIANQLAIAIQQADLFEKLQKSLKQEKEISAMRSRFVSMVSH EFRTPLAIISSSTGILQTFGDRLNAEKKQGHLETIQKTIKYTVQLLDDVL MINSVETEKIEFKPETLDIIDFCRRLIREIQGTSYSHVIDFSLNSTQLIL DHTLFAEFDPKIIRQVLTNLLTNAIKYSPGSSTVSFSLNITDKQIVFIVQ DYGIGISETDQVNLFASFYRGSNVGNISGTGLGLAIVKKCVDQHQGKITL ESKLNQGTIFKVTIPRYNLIGNG 131C_1565 (Bold and underlined region = SEQ ID NO: 20) (SEQ ID NO: 32) MLIPVNAISATELKSAIIYNPLLATADTTVREAVVQMSGIAANPLWLTVP AVCSIPHSANNYLEHLQIQACCSCVLIVENNRPVGIFTQQDVVEISAQRP NLEDLALREVMTHPVITLQESKFTDLFFTLNLLQHHRIRHLPLVDEENQL VGLLTYEILRQILRPVDLLRLRLVHEVMTTNVLCAPANVSILEIARLMTE NQVSSVMIVETQASLTIPLGMVTEHDIVQLKALSVNFDTCLAQTVMSTPV FCVTVDESLWNVQQIMEQRFIQRLAVTGSKGELVGIVTHSSILEALNPWE LYKLTAVLQEKVLQLETEKIQLLGNRTLELEKQIEERTIKLRRKAEQEKL INQIATQIYSSLDLQEILNNTVVGVRSLLNCDRVIVYQFSGDFRGQVIAE AIVAGGHSVLHQEVHDPCISPEWLELYRQGQIRVINDINTESITQCHQQM LKDLDIRGKLLSPLIVENQLWGLMLASYRDIPHNWELEEIELVQQISLRV AIAIQQANIYQQTQIEIHQRQQAEELIKQQLAELKIWKNRYELASTASGQ IMYEYNLLKDAPVWAANMEEVLGYSYSECPRNLAEFMDIVYPEDRDRLYS LIQKNLAQKSPLSTEYRLRRKDGNYIWVEDRNQVVLDDQGEIVVVIGAIV DITVRKNSEEKLSKLFQKSEKLQQRLSLVLKGSNDAWWDWDILDDTIYYS ARWFSLLGYKHEELYLKSESFWENFMHPEDIDPIRGNFNQALDDKNIEFI ESKFRLRHKQEYYIFINCRSYILRDETGKAVRVSGANTDITQLVRKEEEL QATLNQLSQFNQKLEARVQKRTVQLQNLSSRLELAIKAAKIAIWEWDLDN NHTIWDKKMYELYGVNPLEYKDGMEILQTALHPEDAVRVNEILQHKLKDG EEFEMDFRIVLPDGKIRVLQSYGIIKRDSQGKAERVIGVNRDITEWKQAE QKIKQQAEREHLLRETTQRIRQSLDLHTIFNTAAAEIRQLMNADRVGIFK FDPVSNFNYGEFVSESVVPGFISALEMKVHDQCFGEKFSPDYAAGRMQIV DDIDNAGLADCHRDILAQFQVKANLVVPLIQGKNLWGLLCIHQCSQPRHW EDFEIELVQQIANQLAIAIKQSMLYEQVQSELIIRKQAEVEIYLQLQRQR AIQDITQEIRSSLNLNHILTTITAKVQELTQA ERVIVFRLFPDGKSQIVE ESVANGYMTFKDSYWEDEKWSQDILEYYWQGKPRIVLDVMDDIWTDCLKA YSRQGNIRSKIVAPILQDLVENENGRWVNHPHNKLWGVLVVH ACGEKRIW EESEAELLQQIANQLAIAIQQADLFEKLQKSLKQEKEISAMRSRFVSMVS HEFRTPLAIISSSTGILQTFGDRLNAEKKQGHLETIQKTIKYTVQLLDDV LMINSVETEKIEFKPETLDIIDFCRRLIREIQGTSYSHVIDFSLNSTQLI LDHTLFAEFDPKIIRQVLTNLLTNAIKYSPGSSTVSFSLNITDKQIVFIV QDYGIGISETDQVNLFASFYRGSNVGNISGTGLGLAIVKKCVDQHQGKIT LESKLNQGTIFKVTIPRYNLIGNG Pse7429DRAFT_2072 (Bold and underlined region = SEQ ID NO: 13) (SEQ ID NO: 33) MSVAKLNASELSSAIIRPPVVVAAHTTVMEAIAQMLGGGFDTSVQSAPND RHNDRHNDCQESTSSYVIAIAEDGRAIGILTERDVMRLSFQQADFTRLQI HEVMTCPLVTLYEADFCDISLAVQLFQQHSIRHLPILDYRDRPVGIVTAE SLQHFLQQHQQNSAAELTAKNIAREQLIAQIADHIRLSFNLQEVLDSCVQ EVRNFLQCDRVVVYQFQSDWSGFIISESVESPFVISLGNHIQDSCFQSQA KQRYDHDQPIIVNNIYNAGYAPCHIEVLEQYQVKANIVIPLQVSGNLWGL LIGHQCREHRDWQPEDASLLRNIAIHLAIAIQQLYAYEQAQKELTERQRS EALIQQQLAELTEWYYRYEAAEKASGQMLYEYDLSSKSLIWGANIARVLG FTVSESPKNLSDLLSAIHPEDRNHFFQTAEICRTNQTPFFCQYRLKHQEG YYIWVEDRNQWLFDDRGEAKRLIGMIADISDRKNAEINLKISEAHHRALI KAIPDLFMRIDRSGIYLEFVCIPSQHRIIGHLLDMNGVHVSETIPPELAQ RRMEYIELALQTQSLQIYEQDFSTPEIDHIEEVRVVPYHENEVLLLVRDI SDRKKAERELKHTEKLFREAQRIAKIGNWELNLTNQVLYWSDEIFRISEI DPQQFSASYETFLNTVHPEDREMVDRAYQQSVSDRLPYNIVHRLLLPDGR IKYIQNQGETIYAEDGSPKLSQGTIQDITSLKQTELELENLNDQLEARML ERETRYWALMNGASDAIMLADLQGNILEVNMQAEQILGYSRAELTSMHFT QLHPEEELTRTRDAFESLTHQQKIQVYDIIFITKNGQLIPFDVSASVIDI QGEPILQGIFRDIRDRKQIESDLQESRDRSQQKASQETILRKITQRIRQS LNLQVIFDTACHEIRQILQADRVGIFQFDADTNYSDGEFVAESTVEGFSS VLAIRLQDYCFGDSYSFSYSQGRCQIVDDIYQTDLEKCHTCILEQFQVRA NLVIPLLCGEALWGLLCIHQCSAPRHWQNFEIELSQQIANQLGIAIYQAS LYQQAQSELLIRQKAEVAISQQLRQQQTIGAITQKIRESLDINAILSTVT RQVKEVLNC DRVIVFRLFSYGDSQIVEEAVSPEFTSLKSLHWENELWSPA ILDYYWQGKPRIVPDVMVDVWTDCLIPYSIEGQIKSKIVAPILQDLGNIE RSRWISPLANNKLWGVLVVH ACAEKRVWQDSEAQLLQQIANQLAIAIQQA SLFAQVQQELSDRQQAQQQLTATNRKLALSNQELERATRLKDEFLANMSH ELRTPLNAILGITEGLQEEVFGVLNAKQKQVLLAVERSGNHLLDLINDIL DLAKIEAGKVTLDRSLTNIEQLSQSSLMFVMQQALQKNIQLHIQVEKSLP DLKIDERRIRQVLINLLNNAVKFTLENGRVVLEVILHKVNDSNLQDVIHW VRFAVIDTGIGITPHALQTLFQPFIQVDSALNRQYEGTGLGLALVKRIVE MHGGQVKATSDFGVGSCFTIELPYNERDSSLLLKHSNSFPSDFVPEPDAK DSQLGHPLILIAEDNEANIITFSSYLSANGYRVIVAKDGQTAVDLVQSEH PDLVLMDIQMPGMDGLKAIEYIRQHQLSNAPIIAVTALAMVGDRERCLAA GANDYLSKPVKLKKLAEVVQQFLHPPC Lepto7104DRAFT_1016 (Bold and underlined region = SEQ ID NO: 8) (SEQ ID NO: 34) MATPRPADLTAAIIDKPLTVQPDVSAGTAIALMGGVSTPGPTGHDPAGED GLHMEAGSCVVVVEQGRVVGLLTERDVVRLSAQQRSLDRLSVAEVMTQPV ITRRLSDLTDLTSTIELLQQHRLRHLPLVDEQDCLVGLVTHDSLWQAFSP LKYCNLTEALERKVTRLETERLALLENRAAELERQVAERTQMVQVQAERD RLMAGLAAQILASLDVQVILDTTVQQVQQILGCDRASIWRFEADWTTVVV AESNDADRSLIGERIADKCFLETQVEAYRQGRIRVVSDIDAIEMSDCHRN MLIRLQTRAKILVPLLCGDELWGLLNVTEMQPRDWQPAEVEFVRSLSIQL AIALNQASTHEQLRSELQERQQAERQLRQSTERLKKAQRIAHIGNWELDL QHNTSYWSKEVFRIFEVDSQQFAASYEAFLDLVHPDDRTLIDTAYANHLR DRQPFSLVHRLRLADGRIKYVREQCETIYSADGTPRISQGTVQDITPQQE AEIRRDRAETTLRQLTEGTAAVTGEAFFPALVHHISEALGVRYVSISQAM PDGFQVLAFFADGELSVPLFLPYDELPCCFEALQTGSCCHPTGVQALYPD NALFTDLQVDSYLGVRLQNAAGDPIGNICILHDAPLADLDWAKTLLTIFA ARAGAELERLMTAQALEQLNGELESRVVERTAELAERETLLQDFLDNAND LIQMVDVSTGRFEFVNRAWQNVLGYTTAEVAQLTCFDVLAPDCLPHCQTV FTQMQSGSITHVEQMELTFICKSGQRVVVEGNVNCRFAVGADGSQRPVST RGIFRDITDRKTTEQELQRREARYRGLMEGAADAVLLIDLEGNILEANQN AAAMFGYPLAELSTLHFTQLHPAETLPRAAAEFAEVAQGQRTQVLDMPCC RRDGSVVPVDITASVISTGEGRLVHGALRDISDRKRYETALQESQQFLQT VLDTVPLSVFWKDQNSRYLGANQRFLKDASLGSVSELVGKDDSAMPWGVT EADAYRAADRVVMDSGEAKLGIIELQHQQDGAVIWLETNKLPLRNLAGEV VGILGTYRDITERKNAEIALQRQLAAIEAAVNGIAILENERYLYFNSSHA KMFGYEQAEELVGQSWRMLYSPEQLERFDREILPILSAEKSWQGEVTATR KDGTTFPEQLSLTISTDNLLICVCQDISERARLDAERKQAEAALRESERR YAMLAQAVPVAIFRFDLEGHCTYVNERWCEMTGKPIDFALDDRWLETIHP DDRERTQTVIQQWLQTGAVAPFQNEARILRDDGSIIWYYCQMLLETDVNG AMLGYVGTLTDISDRKQSEEALGESEEKFRQLAEVVDAVFWILHLNRTDR VYVSPAYERIWGRPCTDLYITPDAWIDRIHADDREQVLAAIPKQLEGTFD EEYRIVRPDGTQRWIHDRAFPIRNAQGQVYRLAGIAEDITERKNSEEIIC QQAEREVVLREITQHIRESLDLQTIFNTACDEIRAFLRADRVGIFKFYPD SGYDDGEFVAESVVNGFSSAMAIRIHDHCFGENYANLYAQGRYQVVDNIY SNGLTPCHSDILAQFQVQANLVMPLLCNHELWGLLCIHQCDAPRHWQQSE INLGQQLANQLAIAIQQASLYEQVQTELLERQQAEAKIARQLRQQTALEL ILQQIRQSLDLPELLAIATQQVQELLQS DRVIVFQVAQNGHSCILEEAVA PDLPQLKAMQWDDETWSQDILEHYWQGQPRIVPDVMEDHWTDCLVEYSKA GQIQSKIVAPILQELCDIETHRWASPEGSSKLWGVLVVH ACRTRRVWHQQ EAQLLQQIANQLAIAIQQANLFEQLQQELQERQQAEAQLTLTNGELMRAT RLKDEFLANMSHELRIPLNAILGMTEVLQDDDVFGPVNAQQLKALKTVER SGTHLLELINDVLDVAKIEAGQLELDCHPTAIAPLCQSSLAFIKQPALKK GLQLAVKLPPNLPEITLDERRIRQVLINLLSNAVKFTLEGGHITLDVSLL PPTQSHPELSYLRFAVTDTGIGITPENMQRLFKPFVQVDSSLNRQYQGTG LGLALVKRIVELHRGQVGLTSDVGVGSCFTVELPYGAGIPAPPVPAPPSA IGPATPLPKVAATPTTTPLILLVEDNEANISTLRSYLQAKGCRVEVAHNG EEAIDWAQHKTPDLILMDIQMPRMDGLEAIGHLRRIPSLANVPVIALTSL AMAGDRDRCIAAGATDYLTKPVSLKQLNERIHALLTP Lepto7104DRAFT_1307 (Bold and underlined region = SEQ ID NO: 18) (SEQ ID NO: 35) MRRFSWSRHLRQPFLLWWLLLPLGLQTVGTAVLIGVLLHGNAAQPAVESA NPLPAANGYLTPAIALWGAVQVLAVGLGAAIARTVAAPKRPQGGLPNASA SHDCRMIEAALQASEARFQTLMAHIPGMVYRYLPGSDGDGAFTYVSAGCY ELFGLSPNQVLQNANAVWGLIHPDDWPSLQASVASAVARCADWHWEGRFT TVTGQPRWLQGRARPQPTPAGAVWDGLLIDITALKQTETALNQEISYRRA LLNASIDGVVIVDREGNVLEANHSFTAMLGYTPAEILSLNVADFDVDLGH LKEDLKSEKTKLCLDRFERLHRRKDGSTYAVEISANAVDWNGQAVHLCVC RDISDRVRAEAIRRESEARYLSILEEQTEFITRFQPDGKLIFVNNAYCRY FSQSKAQLEGQNYQPVVYPADQPAIDRCLASLSPETPIRTVENRVYVRGE LRWTTWTNKAIYDDCGNLIELQSVGQDIHDRKRAELALAESEARFQRLTA ASPAIIYTVIESLQGIVRFEYLSPAAEEIHEIPIATLMQNGALISEQMHP DDRERYLEAYAASLQSMTTFICEWRIITPSGKTKWLKANSRPEQRPSGEV AWHGITLDITPRKQAEAALGNLQAALLEAQQVAHIGNWEFDLASQKITWS PELFRMFGLDPAQGEPTYADYLELLQPDDRILLQQAVDRAIAEGTPYRLD YRVLLPDGSLRYQEGRGKVERDRTGQWRLFGTALDITDRKHTEIALQAS QLRLQLAINSTGTGTWDWNMQTNEVLFDQKLWRALLGYGADAAIDNSVAE WESRIHPKDKPQVQADIARHIRGETEIYENTHRLRCHDGTYKWNLAQGKI IERDDRGNPIRFVGIHRDVSEQVLLDAGRRLAEEALQASEARFRAIFEQA AVGINQADASGRFIQANQYFCGLLGYTQAELLRLTVQDLTHPEDLERDRL QILRLFQGKQKGFTTIKRYRHRHGSWIWTEVTLSAICNPAGEVISDLAIV VDIRKLRQANAALKASEARLRAIFDQALAGINQIDSQGQFTEANQYFCDL LGYSRDELLALKLEDLIHPDDMERCREPVDRILRGEIDNLRLERRQRHKN GDWIWTEAMISLLRDEAGEVIGNLAVVVDIRERIRLEADRKRAEQTIRQQ AERETMLRKLTQSIHRSLDLQTIFDTACREIRACLQADRVGIFKFRPGSS YSTGELVAEAMVDGVTPVLAIPIHDHCFGERRAAFYAEGHCHIIDDIYAS DLENCYIDFLAQLQVRANLVIPLLCGRDLWGLLCIHQCAGPRHWLRADID LGCQLAHQLALAIKQALFVEQIQSELQVRQRAEAKIAHQLRQQTALGMIL QQVRESLDLDQILATVTQNVQEILQS DRVIIFQVHSDGHSKIVEEAVSES LPTLKGMRWEDEVWSQDILDVYWRGQPRIVADVMADTWTDCLVDYSQAGQ IQSKIVAPILQEIRTSEGHRWVAPRAKNKIWGVLVVH ACRQKRVWQDSEA QLLQQIANQLAIAIQQSTLFEQLQQELSDRQLAQQQLTESNQELAVANQA LSRATRLKDEFLANMSHELRTPLNVILGFAQILSSDLSLQAQQQEYIRIM HRSGDHLLHLINDILDLSKIEANRITLEPESIDLLELLHDLQGMFQERAE DKELRFTLALAPDLPQYIVADPNKLRQVLINLLGNAIKFTQEGSVALRVS LALPEHPEPQPEPPQPYLSFAVEDTGTGIAPAELASIFDAFTQAKAGKVS LEGTGLGLAISRSLVQLMGGSLTVSSRLGQGSTFCFSLPCHRGRAEDVAL TNYPGAVTGLAPAQPNYRILVVDDQPENRQLLLAAFSQVGLAVREAAHGA EAIAQWRQWQPHLIWMDLRMPTLDGCEATRRIRAESAAIANGDRPIIIAL SAQASNDECSNALAAGCDDFVSKPVKLNLLWTKMSDYLGLRYVYAETPTP AGLVNPTSAKAIRIDTSDLQVMPPEWIGALHQAALHCDSHDTAQLIQQIP AEHGALTTSLNRLLDGYKFEVIMQLTQPYLEAAP LEP6406DRAFT_1154 (Bold and underlined region = SEQ ID NO: 9) (SEQ ID NO: 36) MIRAMKVDLTAAIVPSPLTVTPETLVQDAIALMSSVRTLCSTDRNPTSND NLHLEHRSSCVLVVVENDLVAGILTERDVVRLSAQQQPLDQLLVAEVMAQ PVITHRQSDLTDLFSTIHLLKHHHIRHLPVVDDQNRLVGLLTHESLRQLT RPVDLLRLRLVQEVMTADVLCAAPDSAMLEIAQLMADRRVSSVVITLPGG STDAPFRRAVGLLTERDLVQFQALGLSLTTTTAQTVMSSPVFAVAPQDSL WTVQQVMEQHRIRRVIVAGEQGELLGIVTQTSLLQAFNPIELYQLAEVLE QKVVHLETERIALLQSQSAELEWHITESNQAIRMQAEIDRLLQGFALATT HLMTLQDGHESVQAALDALGSALRVDRSYIFENHPHPKTGEMVLSQRWEW VAEGVTRQIDNPELQNIPVDKVLPNWYQSLSQGQTVGGLTKDFPEEEQAH LRPQGIVSILLVPIFIEDYFWGMVGFDDCHEERVWENSTQSALKSIAGTI GSAIARRRAEANATLLAKRLQEAQRLAHVGNWEQDLQRHTFYWSEEVFRI LEIDAQQISASYETFLGLVHPDDLTLVDEAYANHLRSRQPTSLVHRLQMP DGRIKYMQEWWETTYSADGAPLISRGTAQDITQQQEAELCRERAEAALRQ VIEGTAAVTGEAFFPALVRHISAALGVRYVSIDQAMPEGFQVLAFFADGE LSPPLFLPYNELPCCFKSLQTGSCCHPSGVQALYPGNALFHDLQVDSYLG VRLQNAAGDPIGNLCILHDAPLADPDWAQTLLSIFAARAGAELERLMTAQ ALEQLNGELESRVAERTAALAEREALLQDFLDNANDLIQMVEIDTGRFEF VNRAWQTVLGYTTDDVAQLTCFDVLAPDCHPHCQAIFAQMQSGDITHLDP MELTFVGKSGQRVVVEGNVNCRFVTEADGRQRPVSTRGIFRDITARKAAE LELERREARYRALMEGASDAILLANPEGYLIEVNPQAVDLMGYEHHELVG MHFTQLHPPEALSTVSEAFGSLAQGGRIEVLNFEILRQDGQRVPVDITGS VIEVGEETIIQGIFHDIRERLQAEQALRDSEIRFRRVFESNVVGMIFADF SGHISDANDRFLDMLGYSRQELESGCCLNWADLTPSEYQAQDEAVIAHLQ HHEAITPWEKAYRHKDGHLVPVLIGVAVLSREEGSCVGVVVDISDRKRYE IALQESQQFLQTILDTVPLSVFWKDRTSKYLGANQRFLQDADLSSVSELV GKTDLDLPWGATEAEAYRADDRAVIDSGEAKLGIVETLHQKDGAEIWLET NKLPLRNLAGDVIGILGTYQDITERRNADIALQRQLVVIEAAINGIAILQ NERYLYLNSSHVELFGYQSPQELIGQSWRVLYSPEELERFDQEIWPALYE QMSWRGEVMATRKDGTTFPEHLSLTLSPDNLLICVCEDISDRKQTEAALK ESEQRYAMLAQAAPVAIFRFDLQGQCTYVNERWSEMTGKPIASAMGDRWL ETIHPDDRERSQTETQQWLQSGTVTMFQNEARILRDDGSIVWYYCQVLVE TDANGTQTGYVGTLTDISDRMKAEQALRDSEIRFRRVFESNVVGMLFADL SGHVTDANDRFLDLIGYSRADLEAHRINWAQITPPEYVEADQRAIDQLQR YGEILPWEKEYLRPDGRRVAVLISVALLSAIDGRCVCVVVDISDRKRYET ALQDSQQLLQTVLDTVPLSVFWKDRQSVILGCNQPFASASGFAEVADVLG KNNFDLGFTQAEAESYTADDYEVMTSGIAKLGIEETVTPAGSQQRWIETN KLPLRDGAGNAIGIVGTFQDITDRKQAEEALRESEEKFRQLAEVVDAVFW ILHLNRTDRVYVSPAYERIWGRPCTELYVTPDAWVEMIHADDREQVLAAI PKQIQGTFDEEYRIIRPDGTQRWIHDRAFPIRNAQGEIYRLAGIAEDITE RKRSEEVIRQQAARETVLREISQRIRESLDLQTIFDTACEEIRTCLQADR VGIFKFYPNTGYDDGEFVAESVVNGLSSVVAIRVHDHCFGENYSTLYAQG RYQVVDDIYHPGLTSYHADILAQFQVRANLVMPLLCNHELWGLLCIHQCD GPRHWHQSEVDLGQQLANQLAIAIQQAILYEQLQAELQERQRAESTITQQ LRQQTALELILQQIRKSLDLPEILAIATQQVQELLHS DRVIVFQVYHDGH SRIVEEAVTPDLPSLKAMHWEGETWPLDILEHYWQGQPRIVPDVMDDIWT DCLVDYAQAGQIQSKMVAPILQELRSVEEHRWVCPEGSNKLWGVLVVH AC QTQRVWQADEAQLLQQIANQLAIAIQQSNLFEQLQQELTERQQAQHQLTE RNEELIRATRLKDEFLANMSHELRTPLNTILGMTESLQEEDVFGPVNPQQ LKALKSVERSGLHLLELINDVLNVAKIEAGQMELDYTSTEIALLCRSSLT FVKQPAFKKRIQLTVNMPPDLPEITLDERRIRQVLINLLNNAVKFTPEGG HITLDVTPLTPSPPSKEPLYLRFAVTDTGIGITPEDQQRLFQPFVQVDSA LNRQYQGTGLGLALVKRIVELHGGQVGLTSAVGVGSCFTFDLPYGVEIAL LPTPLGPQPDLSATTPLQTEAAIPESKALILLAEDNEASISTMVSYLEAK GYRVAIANNGQAAIEKAQRLRPDLILMDIQMPGMDGLEAISHIRRDPNLA DIPVIALTALAMSGDRDRCLTAGATDYLSKPVRMKQLVKRIQTLLNP LEP6406DRAFT_2712 (Bold and underlined region = SEQ ID NO: 21) (SEQ ID NO: 37) MRQFSDLNRPLGPISLQVLFRVSLGLQTVGTMALVGYLLYGLLGYGGGVG AGLPPLLSPLGGSVPLAIALIILICGTWGVTIVLGFFTSRQITQGIDQVI QASQTLAAGQMPPPLPRGSMIGDLDRLAQSFQQMATAVDLYQVQTQDNLA ALEEKFTLLFHYSPIPTWIATLEEGRCLLVNDSFCQLMGYAQAEIIGQTC RQLQFWDNLVDYQNFRHGLTTQGQVRDFECVFRTQSGGTKTLLLTAQVSC LEGQDCILGIAHDISDRKQAELALRDSEMRLQALLANTPGMIYRYLPIDD GGGTFLEVSAGAYELLGLEPEQVRQDVSTVWALIHPEDVLTLQDSVEIAV RDCTDWHWEGRLTTPSGELKWLRGYSRPYVTPAGIVWDGLFTDITALKQT EISLHQEVSRRRSLFETSIDGIVIVDRAGNVLESNARFANMLGYSLEEVK TLNLVDFDVNLSSVEIEGKIDKDELCLDHFESRHRRKDGSIYAVEISANT INWGDQSVSLCICRDITERKRNELALQTSQLRLELALDSSGTGTWDWNME TNEVFFSEKSWRAMVGYGADDRFGNTITEWESRIHPEDKAQLEVDIAKHL RGETETYESVHRIRCQDGTYKWNLAQGKVIEWDQAGNPVRFIGLYRDISD RKQTEIALSNLRSQLERAQEIAHLGHWSFDLDTQKLTWSDEVFRIFDMTT DQDEPTFREHLEQIHPDDQSSWLERVAEANQGIPQNFCFRILRPTGEVRY VNSYLELEYEGEQIVRMFGVVMDITEQKQNELALQASEARFRAIFEQAAV GINQADVSGQFIEANQYFCDLLGYTRDELLALTFQAITHPDDFQQDSVFS RLLAGELTSVTAQKRYRHKQGDWIWTEVTVSLIHDADGRAISDLAIVLDI SDLKQANAALQASEARFRTIFEQAAAGINQIDASGRFTEANQYYCDLLGY SRAELLTLTFVDVLHPEVLAQYWSENNFILSGEIEFLEYEKRLRHKNGDW IWVKSNISVLRDQAGELAGNLEVVVDIRDRKQAELALHASEDRFRAIFEQ AAAGINQIDVSGRFTEANQYYCNLLGYSRAELLTLTFVDVIHPEDLAKHW SEVDRIVRGEIDFLDYERRERHKNGDWIWIKSNISVLRDGAGQVVGNLAV VVDIRDRKQAELALQESQARFQLLSAASPAVIYTVIETAQGINRFDYISP AAEEIHEIPVDTLLQNGMLISEQMHPEDREHYAATYAASLQALAPFTCEW RIITPSGQTKWLRASSCPEQRPDGDIAWHGIALDISTRKQAELESQTLQT ALVEAQRIAHIGNWAFDLASQKITWSLELFRMFGLDPAQDEPSYPDYLQL IHPDDRLLLQQAIDRAVTAGTPYSIDYQAQLPDGSTRYHEGRGEVERDCS GQITRLLGTCLDITDRKRVEQIILQQARQEALLREIGQRIRQSLDLQTIF DTACQEIRSCLNADRVGIFKFDPDSGYDDGEFIAEACVGGLPSVLTIPVQ DHCFGDNYATLYAQGHYCVIDDIYSANMADCYIDLLAQFQVRATLVMPLF CGDVLWGLLCIHQCNAPRQWQQANIDLGQQLANQLAIAIQQAILYEQLQS ELQERQRAETKISQQLQQQTALGMIWQQIRQSLDLQDILAIVTQQVQVVF QC DRVIVFQLFADGRSQIVEEEVLGSLPALRTMHWEDEVWSQDILALYWQG QPRIVPDVMDDIWTDCLVEYAQAGQIKSKIVAPILQQGHTATGNRWQDPN HPHKLWGVLVVHACH ERRTWKAEDAQLLQQIANQLAIAIRQAHLFEQLQ QELIQRQQAQQQLVERNQELAIANQDLSRATRLKDEFLANMSHELRTPLN VILGFAQVLNSDLSLQPQHQDYIRIMHRSGDHLLHLINDILDLSKIEANR ITLEPESIDLFSLLHDLQAMFQERATDKELQFTLALPPDLPQYIVADPNK LRQVLINLLNNAIKFTQQGQVILSVRLQGAEADQQFHLSSSITSSDTPPT PSLCFQVIDTGVGIPSEEIDIIFDAFTQARAGKSTLGSTGLGLAISRSLV KLMGGELTVNSAPDQGSTFQFAIPLHLARGEDVTSEGSLGTVIGLAPGQS PYRILVVDDQPDNRQLLVTVFSQIGLEVQEAASGADAIAANQQWHPHLIW MDLRMPDMDGCEATRQIRAQAQELDSENRPEDPVIIAFTAQASMDERTRA LESGCDDFVSKPIQLNLILSKMADYLDLRYEYAQTVTPAPGAQSATATAI TLDAQSLRIMPLEWIAALHKAALHCDDQAASSLVQEIPTSQSVLVEGLNR LIYDYKFESIAQLTSPLLLE cya56DRAFT2_02270 (Bold and underlined region = SEQ ID NO: 10) (SEQ ID NO: 38) MLFPPDRLDEEPQILARLMRGERVEHFETVRISKEGKSIEVSATISLLKN AAGEVVGVSKILRDISDRKQAEKSLQESQQFIQTVIDTVPLPLFWKDRSS VFLGCNQQFVRILGAPSSKEVVGKTDFDLLPTEEEASAFQADDRGVMESG QAKLGIEEMLTFANGEQRWLETHKAPLRDWSGNVIGMVGTFQDVTDRKQA ELELQKNTERLVFALKSGAKEFEMQLQQTTDRLSLALNSGAIGYWEWDIQ QNILVWDDRMYELYGYLKENYSHLPYEIWANAVHPDDRDLTETLLQQAVL GKTEYDCEFRIIHPDHSIHFIKAYGTLNRDASGNPLSIIGINFDITDRKQ AEQIILQQANRETLLRGITQRIRQYLDLSIIFDTACQEIQQLLQSDRVGI FKFYPESNFDDGEFVAESVVNGFSSAMEVHIHDHCFGEGYAAEYAQGRMQ VVNDIDNAGLMDCHRDVLAQFQVRANLAIPLLCGNNLWGLLSIHQCAHTR QWQEDEINLIQQIANQLAIAIQQASLYEQLQEELLIRQQSQSKIAQQLRE QQTLATITNKIRESLSIKEILAVVTQQVIDVLSG DRAIIFQLFDNGNSQI VEESVHSNFLNLKALNWDNEVWSQEILDCYWQGKPRIVPDVMNDIWTECL VEYSLKGQIKSKIVAPILLESHISENHRWVATDGYKKLWGVLVVY ACAEQ REWQDSEAQLLQQVANQLAIAIQQASIYEESQQEIAERKQAEQQLTETNQ QLARATRLKDEFLANMSHELRTPLNSILGMNEALQEEVFGGINERQLKAL QTIESSSRHLLALINDILDVAKIESGQVTLELTATDIDSLCKSSLAFIKQ QALTKRIQLIPRIPKHLPKIMLDERRIRQVLINLLNNAVKFTLEGGTITL EVSQVQLESSTTNPTPLKYLKIAVIDTGIGISAENIQKLFQPFIQIDSAL NRQYNGTGLGLALVKRLVEIHGGTVELTSELGVGSCFAINLPINIVSPAI EEQTEQDLSGQSQIGQSQTEGLISPLILLAEDNEANIATFSSYLEAMGYR ILSATDGQQAIDLAKAEHPDLILMDIQMPVMDGLEAIKQIRLDPNLADIP IIALTALAMEGDRERCLAVGANEYLSKPIKLKALADTIRNILKNRN Ga0039498_104087 (Bold and underlined region = SEQ ID NO: 11) (SEQ ID NO: 39) VVESSDDAIITKTIEGIITSWNPAAERLFGYSEAEAIGQPISMLFPPDRL DEEPQIFARLMRGERVEHFETVRISKEGKSIEVSATISLLKNAAGEVVGV SKILRDISDRKQAEKSLQESQQFIQTVIDTVPLPLFWKDRSSVFLGCNQQ FVRILGAPSSKEVVGKTDFDLLPTEEEASAFQADDRGVMESGQAKLGIEE MLTFANGEQRWLETHKAPLRDWSGNVIGMVGTFQDVTDRKQAELELQKNT ERLVFALKSGAIGWWEWDLQSDIAVWDDRVYELYGVSNQTNPQPTYEIWK NALHPHDAEAIEAINRKIAAGQIDEYDTEFRVVHPDGSIHFLKAYGMLKR DADGKPQSITGINFDVSDRKEFEVQLQQTTDRLSLALKSGAIGCWEWDIQ QDFLVWDDRMYELYGYLKENYSHLPYEIWANAVHPDDRNATETLLQKAIL GQAEYDYEFRVIHPDRSVHFIKAYGKVKQDSQGNAESMIGINFDISDRKQ AEQIILQQANRETLLRAITQRIRQSLDLSIIFDTACQEIQQLLQSDRVGI FKFYPESNFDDGEFVAESVVDGFTSAMEVHIHDHCFGEGYAAAYAQGRIQ VLNDIDNAGLMDCHRDVLAEFQVRANLVIPLLCGNNLWGLVCIHQCAHTR QWQEHEINLIQQIANQLAIAIQQASLYEQLQEELLIRQQSQSKIAQQLRE QQTLATITNKIRESLSIKEILAVVTQQVKDMLSG DRAIIFQLFDNGNSQI VEESVHSNFLNLKALNWDNEVWSQEILDCYWQGKPRIVPDVMNDIWTECL VEYSLKGQIKSKIVAPILLESHISENHRWVATDGYKKLWGVLVVH ACAEQ REWQDSEAQLLQQIANQLAIAIQQANLYEQSQQEIAERKQAEQQLTETNQ QLARATRLKDEFLANMSHELRTPLNSILGMNEALQEEVFGGINERQLKAL QTIESSSRHLLALINDILDVAKIESGQVTLELTATDLDSLCQSSLAFIKQ QALAKRIKLIPRIPKHLPEIMLDERRIRQVLINLLNNAVKFTLEGGTITL EVSQVQRESSTTNPTPLNYLKIAVIDTGIGISAENIQKLFQPFIQIDSAL NRQYNGTGLGLALVKRLVEIHGGTVELTSELGVGSCFAINLPINVGFPAI EEQTEQDLSGQSQIGQSQTEGLISPLILLAEDNEANIVTFSSYLEAKGYR ILLANDGQQAIDLAKAEHPDLILMDIQMPVMDGLEAIKQIRLDPNLADIP IIALTALVMEGDHERCLAVGANEYLSKPIKLKQLATIIQQILVRT Ga0039499_10213 (Bold and underlined region = SEQ ID NO: 22) (SEQ ID NO: 40) VVESSDDAIITKTIEGIITSWNPAAERLFGYSEAEAIGQPISMLFPPDRL DEEPQIFARLMRGERVEHFETVRISKEGKSIEVSATISLLKNAAGEVVGV SKILRDISDRKQAEKSLQESQQFIQTVIDTVPLPLFWKDRSSVFLGCNQQ FVRILGAPSSKEVVGKTDFDLLPTEEEASAFQADDRGVMESGQAKLGIEE MLTFANGEQRWLETHKAPLRDWSGNVIGMVGTFQDVTDRKQAELELQKNT ERLVFALKSGAIGWWEWDLQSDIAVWDDRVYELYGVSNQTNPQPTYEIWK NALHPHDAEAIEAINRKIAAGQIDEYDTEFRVVHPDGSIHFLKAYGMLKR DADGKPQSITGINFDVSDRKEFEVQLQQTTDRLSLALKSGAIGCWEWDIQ QDFLVWDDRMYELYGYLKENYSHLPYEIWANAVHPDDRNATETLLQKAIL GQAEYDYEFRVIHPDRSVHFIKAYGKVKQDSQGNAESMIGINFDISDRKQ AEQIILQQANRETLLRAITQRIRQSLDLSIIFDTACQEIQQLLQSDRVGI FKFYPESNFDDGEFVAESVVDGFTSAMEVHIHDHCFGEGYAAAYAQGRIQ VLNDIDNAGLMDCHRDVLAEFQVRANLVIPLLCGNNLWGLVCIHQCAHTR QWQEHEINLIQQIANQLAIAIQQASLYEQLQEELLIRQQSQSKIAQQLRE QQTLATITNKIRESLSIKEILAVVTQQVKDMLSG DRAIIFQLFDNGNSQI VEESVHSNFLNLKALNWDNEVWSQEILDCYWQGKPRIVPDVMNDIWTECL VEYSLKGQIKSKIVAPILLESHISENHRWVATDGYKKLWGVLVVH ACAEQ REWQDSEAQLLQQIANQLAIAIQQANLYEQSQQEIAERKQAEQQLTETNQ QLARATRLKDEFLANMSHELRTPLNSILGMNEALQEEVFGGINERQLKAL QTIESSSRHLLALINDILDVAKIESGQVTLELTATDIDSLCKSSLAFIKQ QALTKRIQLIPRIPKHLPKIMLDERRIRQVLINLLNNAVKFTLEGGTITL EVSQVQLESSTTNPTPLKYLKIAVIDTGIGISAENIQKLFQPFIQIDSAL NRQYNGTGLGLALVKRLVEIHGGTVELTSELGVGSCFAINLPINIVSPAI EEQTEQDLSGQSQIGQSQTEGLISPLILLAEDNEANIATFSSYLEAKGYR ILSATDGQQAIDLVKAEHPDLILMDIQMPVMDGLEAIKQIRLDPNLADIP IIALTALAMEGDHERCLAVGANEYLSKPIKLKALADTIRNILKNRN Cal6303_3693 (Bold and underlined region = SEQ ID NO: 15) (SEQ ID NO: 41) MFNNTTVLTTSELKSAIVRDPLIVKPDMTLIDAIAQMSGVRTLCETTQTI DGQLDNLYLEARASCVLIVEEGKLLGIFTERDVVRLSAQQYSFENLKIRE VMTHPVISLRESDFTDLFFPVNLLQQHHIRHIPILDQQDQVVGLLTNESL RQSSRPVDLLRLRLVYEVMTKEVICGAPDSSMLAIAQLMAKHRVSSIIIV QPDNSETESLQIPVGIITERDIVQFQALGLNLKTCLAKVVMSTPIFAVKP NDSLWLVQQLMEQRLIHRLAVTGEQGELLGIVTQTSLLKALNPLEIYKLA EVLEKKVVKLEAEKIALLETRTVELEQQIEARTFVIKAKAERERLVLEIA TQIRSSLSLQTILDTTVAEVRQLLGCDRVNIWQFDANWQTITVAESTDSP MSLLGKRVIDTCFQDDYAKIYRQGRICVMRDIYKAKISDYHRDMLIRLQT RAKILVPLFCGEQLWGLLNVTESQHPRNWEAEEIELLEALSVQLAIALQQ ATNHQKLQEELHERQRIELILQKLVTGTATVTGEDFFPALVRHIAEALNV RYAVVTEIVDNKLHTLGFWANGALKPSMSYCAVDNACEYSLRDGEFYCQS KVQELFPEDLNLAAMEADSYVGIALKDDLGNAIGNLCILDTQPLTEAQRI EAIAILQVFAARATAELQRQTANNALHRLNQNLEQRVEERTEQLQAREAK IAQQLRLQKTLGVIIQKIRESLDISEILVTVTHQVKELLQS DRVIVFRLL GDGRSQIVQEAVSNEFPVLKDRQWENEVWSQEILPGYWQGKPRIVPDVMN DIWTECLVEYSREGKIQSKIVAPILQDLYSGERDLTVERGGLLPLREKHR WVAPYLTNKLWGVLVVH ACEEKRVWKDSEAELLQQIANQLAIAIQQASLF EQLQQELAERQQAEAKLTDSNQQLAVSNQQLARVTRLKDEFLANMSHELR TPLNAILGMTEGLQEQVFGVVNEQQLKALQLVERSGLHLLELINDILDVA KIEAGQIELDYAPTSVAHLCESSLVFIKQQALQKRIQLEIKLQINLPDLF VDERRIRQVLINLLNNAVKFTPERGCITLEVTQITLNISDADSPEQYFLR FAVRDTGIGISPENIKNLFQPFVQIDSALNRQYTGTGLGLALVKRIIELH GGLVGLTSELGVGSCFTIDLPFAPNHTSPSVIAAGDQPVATSELDPSSPN EVWLTPLILLAEDNEANISTVSSYLKAKGYRIVLAQNGQEAIDVAKTHH PDLILMDIQMPGMDGLEAMRQIRLDPNLAEIPIVALTALAMTGDRDRCLT AGANDYLSKPIKLKQLANTIQQLTNAVKDNK fdiDRAFT29700 (Bold and underlined region = SEQ ID NO: 12) (SEQ ID NO: 42) MFKHTTALTSSELKSAIVRNPLIVGLDTLVIDAIALMSGVRAVCDANKLD ELDIDARSSCVLVVDNHSLLGIFTEKDVVRLCAQQRPLENLAIREVMIHP VIALHESDLTDVFFAVNLLQQYHIRHLPILDEQDLVVGLLTNETLRQSSR AINLLRLRLAFEVMSREVICAAPDSSILAIAQLMTAHRVSSVMIVQPGGS EAAPVKIPVGILTERDIVQFHALGLNLETCCAHMVMSTPIFAVKPEDSLL VVQQIMEQRLIRRLAVTGEQGELLGILTQSSLLQALNPLELYKLAEVLEK KVVQLETEKVQLLEARTAELEQQVEARTTALKTKAEQAQLVSDIAMQIRS SLSLQTILETTVQQVRQFLGCDRVIILRFEEDGPAAVVAESTNSSLSLMG RWIKDGCFQKNYRENYCQGQIRVVKDIYTTQMTNCHRQMLISLQIRAKIL IPLLCNGELWGLLNVSESDKAREWQQSEVELLQALSVHLEIALQQATIHQ QLQEQLRDRQRAEMTLQKLVTGTAAVTGDDFFPALVSHIAEALNVCCALV NELVGDKLYSLGFWENGALQPAISYHIAQTPCEHSLRDGEFYCQSQLQTI FPDNLALQTMQADSYLGIALKDNLGNTIGNLCILDRQPLSQTKYTEAIAI LQVFAARAAAELQRIAANDALHRLNQDLEARVEQRTEELQAREVELHKTS ERLALSLKSGGIGCWEWDILQNTILWDERMSELYGVTPQSDSCIVYDTWT KKLHPDDRTQTETLLQQAVLGQAEYNTEFRVVHPDGSIYFIKAYGVVVRD EQGSPQKMIGVHFDISDRKRAEIALQSSELRFRRIFDSNVVGMLFADFKG DITDANDRFLQMVGYTREELNAGALSWKAITPSEYVFADVGALKHLSQYG AMNPWEKEYYRKDGSKIPVLLGVAMLPGSDYQTICVVVDISEQKAALQER QQAEMQLQQQARHKQLLWNITQTIRQSLDIEVIINAAVTEIRQVLGVDRV ALYRFRADWSGEFVAESVAANWVKLVGSQVKKVWEDTYLQETQGGRFQNY ETLVVADIDQAGLQPCHIELLQQFQAKAYVITPIFVNESLWGLFAMYHNH RPHSWTTWEIELLRQIANQLAIAIQQASLYEKNQSELLVRQQAEARIALQ LRRQQTLGAIIEQIRKSLDLNEILATVTQQVKDLMHC DRVIVFRLFADGR SKIAEEAVSSEFVSLKNRHWGNEIWSQEILDFYWQGKPRIVPDVMNDLWT HCLVEYSQEGQIQSKIVAPILQEVRDQNHRWVSPWATNKLWGILVVH ACQ ERRVWKNSEAQILQQIANQLAIAIQQASLFEQLQQELAERQQAEAKLTEI NQQLAFSNEELARATRLKDEFLANMSHELRTPLNAILGMTEGLQDEVFGS INQQQLKALDTIERSGSHLLELINDILDVAKIEAGQIKLDYTSISVANLC QSSLAFIKQQALQKRIQLETKIPQNLPHLLVDERRIRQVLINLLNNAVKF TPEGGRITLEVNQLSPDTTNNSLRQHFLQIAVKDTGIGIAPENINKLFKP FIQIDSALNRQYAGTGLGLALVKRIVELHGGRVGLSSELGVGSCFTIELP YTPVFPVVEDTQPDVTPEFVSSNLDHAGPLILLAEDNEANISTVSSYLKA KGYRILLANNGKEAIELATTQYPNLILMDIQMPLMDGLEAIKLIRLDPNL VNTPIVALTALAMNGDRDRCIAAGANDYLSKPVKLKQLATTIQQLLST Osc7112_5903 (Bold and underlined region = SEQ ID NO: 14) (SEQ ID NO: 43) MFMRTTALTPIELRTAIVREPLVVSPDTTVMDAIAQMSGVRSLCNTPRTA DGQLDDLHLEARSSCVLVVENEQLVGVLTERDVVRLSAQQRCLENVAMRE VMAHPVVTLRESAFTDLFLAINLLREHHIRHLPILDELDRLVGLVTHESL RQTSRPIDLLRLRTVAEVMTREVICAAPDSSLLTIVQLMAEHRVSSVMIV HPGGISTEPLQIPVGILTERDIVQFQALGLNLETCLAQAVMSTPIFAVRS DDSLWTVQQIVEQRSIRRLAVTGELGELLGIVTQTSLLQALSPLELYKLV QKWEEKVVRLEAEKVALLANRNVELEQQVAARTAALKAKADREQLLNTIA EQIRSSLNLSDILHTTVQEIHSLLGCDRVIIYQFQSELSGTVIAEAITDT GRSVLYREARDPCMSPEWLEPYRQGRIRVINDIYDAEMTQCHQEMLVGFD IRAKLMVPIVIEQQLRGLTIASYRAAPHSWTTDEIELLRQVSLQVAIALG QAAIQQKLQNELVKRQRIEATLIESEQRYAALAAAAPVGIFRTDAEGLCT YVNDRYFQIGGLRPGGTIGQGWQQGIHPDDRDLVIAQWEQFIQGNDSFEL EYRFQRPDGTVTWVYGQCVAELDANGNRSGYIGTITDISDRKRTEVRLQE SEERYASLVAAVPVGIFRADALGKCIYVNHWWCQISGLTPKTAVGEGWKQ GLHPDDRDWVMAECEQSLQRNRSFQLEYRLQRPDGAVAWVYGQSVPELDA DGQVVGYVGTTTDISDRKQAEQKLQQLNQQLETKVAERTQELWQVNSLQR AILDCADYSIISTDPTGIIQTFNAAAERMLGYSAREIIGKATPLLIHDAN EVIDRASSLSAELGQNIPPTFEVFVAKARQAPVSEEEWSYIRKDGSRFPV SLSISTLKDVNQQIIGFLGIAKDISDRKRAELELQKLSDRLALSLKSGAI GCWDFDLVQNTIFWDERMYELYGVTKQSDSPLPYDIWANRLHPEDRTATE TLLQQAVLGQANFETEFRVLHPDGSLHFIKTFGVLVRDARGNPQSMIGVN LDISRRKQAELQRQQLIQELSAFKQALDQSAIVVITDREGVISYVNDRFC AVSGYSRDRLIGQTHRIVNSGYHPPAFFQDLWDTINSSQIWRGEICNRAK NGSLYWVATTIVPFLDEQGRPFQYLAIRFDITDRKLAEATLQQENTFRQQ IVENMVQGLCVFHQFEEFPFVSFTVWNQQMQTITGYTLEEINRLGWYQTL YPNLEDREQAIANCRQMQPIAVEREIQRQDGQRRTISISTSVLSGDDGHL YSLALIQDITDRQQTERENRLLKERLEFLLASSPAMIYSCKPYGDYDATF MSKNIEAILGYKAEEFLSESGFWANHIHPEDAPRVFAHISDLFEHNTHQH EYRFLHRDGHYVWLRDELRLLRDEAGKPIEIVGYFADISDVKQTEETLKI QLAAIEAAIDGIAIIQGDTYLYLNQAHLELFGYERPEEVSGKSWKLLYSQ QELERFEREVFPVLGRDRAWQGEAIALRKDGSTFAEGLSLTLTDDGLLIC VCRDISDRKQIEAELAESEAKFRRLVEGVNDLIWSCEPDGILTYVSPQFK TMFGWEEGAWIGKSFIYLVHPDDRPLVVTGYRKNIKFGKKSSDYEFRHRH RDGNYVWVRSSATPVMNAEGELISIQGILSDISDRKQAELARESSEIRFR RVFESSVSGMMFADFQGNITDANDRFLQMVGYTREELNAGMIHWDAMTPP EYLPADFLAFERLRQDGEIESLEKEYYRKDGSRISVLLGAALLPGSEDQT ICVLVDISDRKQAQKALQESQQFLQTVLDTIPLSVFWKNRESVFLGCNQQ FATTLGLQSTSESIGKRDLDICQEEVEANEYCAMDRRLMETGEAILGIEE TLTLPNGKPIFIETHKAPLRDCSGNVIGLVGTFQDITDRKEAELRLQQQA KQERLLGAITKRMRSSLHLDEILNSTVEEIHQILQSDRTLVYRVFPEGTG TAIAESVSPNRLKLLDILFPEEVFPEENYERYIEGRVYALNDSEDANESI VPCLVEFLADIQVRAKLVVPIIQNQSLWGLLIVHQCDRPRQWQEWEINLL KQIANQLAIAIQQSYLYEQVQSELAIRKQTEKAIALQLQRQRTLGEIAQQ IRESLDINEILATVTQQVKEILQG DRIVVFRLFGDGRSQIVEEAVSSEFP ALKDHHWEDELWSPEILNRYWQGKPRIVPDVMTDIWTDCLVEYATVCQVQ SKIVAPILQEVRSSESHRWVAPGQTKKLWGVLVVH ACREQRVWQESEAQL LQQIANQLAIAIQQASLFKQLQQELTERQQAQQQLTERNEQLAVSNEELA RATRLKDEFLANMSHELRTPLNAILGMSEGLQEQVFGIINEEQIKALQTI ERSSSHLLELINDILDVAKIESGQMELDCTPVSINHLCQSSLAFIKQQAL QKRIQLEIKVPLNLPDLLIDERRMRQVLINLLNNAVKFTPNGGRITLEVS SQQRRADPDSADSPPHFLVKETLRISVIDTGIGIAPEHINKLFQPFIQID GALNRQYTGTGLGLALVKRIVELHGGQVLLTSTVGVGSCFTIDLPCTGCA PSSVDVESQTEPRIEPSGPEQQGGSPLILLAEDNEANISTVSSYLRAKGY RILLAKDGEEAVALAKSENPNLILMDIQMPGMDGLEAMQQIRCDPNLVDL PIVALTALAMTGDRDRCLAAGANDYLTKPVKLKQLASTIQQLLAK Mvag_PCC9802_DRAFT2_00054240 (SEQ ID NO: 44) MFMRTTALTPIELRTAIVREPLVVSPDTTVMDAIAQMSGVRSLCNTTRTA DGQLDDLHLEARSSCVLVVENEQLVGVLTERDVVRLSAQQRSLENLVLRE VMAHPVVTLRESAFTDLFFAINLLQQHHIRHLPILDDLDRLVGLVTHESL RQTSRPIDLLRLRMVAEVMTREVICAAPDSSLLAIAQLMAENRVSSVVIV HPGGISTEPLQIPVGILTERDIVQFQTLGLNLETCLAQAVMSTPIFAVRP DDSLWTVQEIVEQRSIRRLAVTGELGELLGIVTQTSLLQALNPLELYKLV QKWEEKVVRLEAEKVALLANRNVELEQQVEARTAALKAKADREQLLNTIA EQIRSSLNLSDILQTTVQEIHSLLGCDRVIIYQFRSDFSGTVIAEAITDT GRSVLHREAHDPCMSPEWLEPYRQGRIRIINDIYGEPMTQCHQEMLVGFD IRAKLMVPIVIEEQLRGLMIASYRASAHSWTTDEIELLRQVSLQVAIALG QAMIQQKLQNELVKRQRIEATLIESEQRYAALAAAAPVGIFRTDATGLCT YVNDRYFQISGLTPGATIGHGWQQGVHPDDRDWVMVEWKQFIQGNRSFEL EYRFQCPDGTVTWVYGQCVAELDANGHRSGYIGTITDISARKRTEVCLQE SEERYATLVAAAPVGIFRADAVGNCIYVNDRWCQISGLTPKTAVGEGWQQ GLHPDDRDCVIAEWEQSVQRNRPFQLEYRFQRPDGGVTSVYGQSVAERDA DGQVVGYVGTTTDITDRKQAEQKLQQLNQQLETKVAERTQELWQVNSLQR AILDCADYSIISSDPSGIIQTLNAAGERMLGYSAQEIIGQATPALIHDAN EVIDRAASLSAELGQNIPPGFEVFVAKARQGLVSEEEWSYIRKDGSRFPV SLSITALKDVHQQIIGFLGIAKDISDRKRAEAELQKLSERLALSLKSGAI ASWEWNLGQNTILGDERMYELFAVTKPSDACQVYDFWANRLHPDDRIPTE TLLHQAVLGQAEYDTEYRIVHPDGSLHFIKAYGVVVRDAQSNPQSMIGVN FDISDRKQAELQRQQLIQELSAFKQALDQSAIVVITDREGVISYVNDRFC VVSGYSRDRLIGQTHRLVNSGYHPPAFFQDLWRTINSSQIWRGEICNLAK NGSLYWVATTIVPFLDEQGRPFQYLAIGFDITDRKLAEATLQQENTFRQQ IVENMAEGLCVFHQVEEFPFVRFTVWNQQMQAITGYTLEEINRLGWYQTL YPNLEDREQAIANCRQMQPIAVEREIQRQDGQRRTISISTSVLSGDDGHL YALALIQDITHRQQTERENRLLKERLEFLLASSPAMIYSCKPYGDYELTF MSKNMSAILGYKPEEFLSESGFWANHLHPEDAPRVFADLSALFEYNTHQH EYRFLHHDGHYVWLRDELRVVRDEEGCPTEIIGYFADISDVKQTEETLKI QLAAIEAAIDGIAIMQGDTYLYLNQAHLELFGYEHPQELLGKTWQLLYSP EELERFEREVFPVLGRDRAWQGEAIGTRKDGSTFAEGLSLTLTENGLLIC VCRDISDRKQIEAELAESEAKFRRLVEGANDLIWSCEPDGILTYVSPQFK TMFGWDESAWIGKSFIYLVHPDDRSLVVTDYRENIKSGKKSSDYEFRHRH RDGNYVWVRSSATPVINAEGELISIQGILSDISDRKEAEIARESSEIRFR RVFESSVSGMIFADFQGNIIDANDRFLQMVGYTREELDAGLIHWDAMTPP EYFPADVLAMERVMQDGAIEPWEKEYYRKDGSRISVLIGVALLPDSDDQT ICVLVDISERKQAQKALQESQQFLQTVLDTIPLAVFWKNRESVFLGCNQQ FAQTLGLPSTTESIGKKDLDICQEEVEANEYCAMDRRLMETGEAILGIEE TLTLPNGKLIFIETHKAPLRDCSDNVIGLVGTFQDITDRKEAEQKLQQQA KQERLLGAITKRMRSSLNLDEILNSTVEEIHQLLQSDRTLVYRVFPEGTG AAIAESVSPNRLKLLDILFPEEVFPEDTYERYIQGRVYALNDSEDENESI VPCLVEFLADIEVRAKLVVPIIQNQTLWGLLIVHQCDRPRQWQDWEINLL KQIANQLAIAIQQSYLYEQVQSELAIRKQTENVIALQLQRQRTLGAIAQQ IRESLDINQILAAVTQQVKEILQG DRIIVFRLFGDGRSQIVEEAVSSEFPALKDHHWEDERWSQEILNRYWQG KPRIVPNVMTDIWTDCLVEYASVGQVQSKIVAPILQEVRSSESHRWIAPG QTKKLWGVLVVH ACREQRVWQESEAQL LQQIANQLAIAIQQASLFKQLQQELTERQQAQQQLTERNQQLGASNEELA RATRLKDEFLANMSHELRTPLNAILGMSEGLQEQVFGIVNEQQIKALQTI ERSSSHLLELINDILDVAKIESGQMELDCTPVSINHLCQSSLAFIKQQAL QKRIQLEIQMPLNLPDLLIDERRMRQVLINLLNNAVKFTPNGGRITLEVS RQQRPADPDSADSPPHFLVKETLRIAVIDTGIGIAPEHINKLFQPFIQID GALNRQYTGTGLGLALVKRIVELHGGQVGLTSTVGVGSCFTIDLPCTACA PSSVYLESQTEPRIEPSQPEEGGSPLILLAEDNEANITTISSYLRAKGYR ILLAKNGEEAIALAKSENPNLILMDIQMPGMDGLEAMQRIRSDPNLVDLP IIALTALAMTGDRDRCLAAGANDYLTKPVKLKQLASTIQQLLASK Cyan7425_1390 (Bold and underlined region = SEQ ID NO: 17) (SEQ ID NO: 45) MPTQKVLESAIVSNPLIVLPETTVIDAIAQMSRAQITGSALSITATNEVH QPAHSSCVLIVADCQLIGIFTAADVLRLIVQQRLQEGLLIREVMTHPVIT LPGVAFTDLSVAINLLQQHRIRHLPLVDSANYPVGLLTYETLLATQNTVL LEAATLEPELQVAARSTARKLEVEWEKLVAEVASKIRSSLSLSTILNTTV EQVRQVLGCERVNIWQFETDSQIAVVAESTDFSISLIGEQVIDNCFQRGK AERYRQGSIRVVSDIYTTEMSDCHRQLLTRLRTRAKILVPLICGRTLWGF LNASESNQRRDWQPAEIELLQTLSLHLSIALQQATTHQRLQKELLARKQV EACLRDREQRYGSLISTAPVGFFWTDAEGECIYANDRWCEIAGLSLEAAE GQGWQAAIHPEDRERVRAEWQQAIQESRPFQLEYRFQRPDGAVIWVYGQV VAEKNDMGAIGGYVGTITDIHARKQAEQQLHNLIAGTAAATGQDFFPVLV QHIAQALNVPYVLVTEKIGGDRLCTLAYWANGELKPTLSLPIANTPCSHV LQDGKFYCASQIQQQFANTLEGIELGAESYLGIALRDSQGEAIGTLCIVD HQPIQEPQRLENLLVAFAARAAAELERERATQTLAQLNRELETKVAERTA ALKASEERWQLVLKGANDGIWDWDLTTNRVFFSERWKNMRGLNQEQVSDR LEEWSRSIHPDDYNCVMANLEAHLAGQTEFFEQEYRVRCQDGSYIWVLAR GQALRDSSGQVVRMAGSEIDITARKQAEQENLRLKERLQFLLSVNPAVIF TSEPGEDYAITFISDNVQTLMGYTPGDFITHPRFWADRIYPEDAPRIFAG LSRLFEQGYHTHEYRFLYQDGFYHWVRNELRLFCDPAGHPLEIVGYCADI SDLKQVEMELAESEAQFRCMVEGVNDLIWSVNDQNRFTYLSPQFATLFGW EGREWIGHFARELIHPDDHPKLADYTQQVMEGRSLDNLEFRHRHQDGHFV WVRSSATPLISSTGNVIGAQGILSDITTLKQAEMALQQSENRFRRVFSSN VVGMMFTDFSGAIFDANDRFLAMVGYSRAELQAGELNWVTLTPLEYVQWD IQAMLHLEKYGSIEPWEKEYYRADGSRIAVLIGVALLSETGSSCVCVVMD ISDRKHAEQTIQQQIQKETLLRELTQRIRQSLDLQTIFTTACQEIRQVLQ ADRVGIFQFYPTSNHNDGEFVAESVVEGLPSVLATPLHDHCFGEQYAPLY VQGRYVAMEDISQLDPCHTDLLNQFQVKANLVIPLISGNDLWGLLCIHQC RSTRRWQATEIDLSQQLATQLAIAFQQAVLYKQTQLELQERQLAETTIAQ QLRQQKNLGTIIQHIRESLDLQQILATVTQQVKEALQG DRVIVFQLFPNG KSRIVEEAVSSGLTVLKAGHWEDEVWPQEILPYYWQGQPRIVADVMDDRW TDCLVGYSKQGEIVSKIVAPILQDIHTFEENPWANPSKRHQLWGVLVIH A CRQPRVWKAEEAQLLQQIANQLAIAIQQANLFEQLQQELTERQQTQQQLT ERNQQLAESNQKLAHATRLKDEFLANMSHELRTPLNAILGMTEGLTDVIF GSINTQQKKALQTIDRSAHHLLELINDILDVAKIESGQIELNCAATSVLL LCQSSLSFIKQQALRKNIHLEVQIPPHVPDVWVDERRIRQVLINLLNNAV KFTPEGGSVTLSVQRQLIVQDPPPLQGITKVRVHRTPIEQQLGIQLQTSQ FEVHNYLRIAVTDTGIGIPSHYLHKLFQPFVQIDSALNRQYTGTGLGLAL VKRIVELHGGEVGVTSTEGAGSCFTIDLPCVSGSSSSSSPFLAESSPAHL SDPANPPCILLAEDNEANISTISSYLKAKGYRVLVAKNGQEAIDLGQAAQ PDLILMDIQMPGVDGLSAIQQLRQAPSSAHLPIIALTALAMNGDRDRCLA AGANEYLSKPVKLSQLVILIQQLLTQS Cyan7822_4053 (Bold and underlined region = SEQ ID NO: 5) (SEQ ID NO: 46) MIIPFPQLTPAIVRNPLVLSPDTKVLEAITSLINQRSQPVKSNCAVVVEN GQIVGIVTKGDILVALAQSQTLDFLTISQVMSSPVVMLRESEFTGLESAI NLFQTHSIDHLPIIDSENHLVGLLTSDSLSAVIQSYIMKDQKIAEKKTTL QLENSFQAAILDEINHISSPKQEQRKLQESELNYTSLAEIAPIGIFRTDT QGYCVYVNPRWCEIAGLTSEEAKGKGWEQVLHPDDDDEVSAQWYRSVEEN RLFQLEYRFKRPNGEIRYVYGQSVALRDINQQIIGYLGTITDITEQKKTE YRLKEALRLAKLGNWELDVQNNIGYWSEEVFHIFGREPQPFSPSFDGFLE LVHPDDRSKVVASYTQHLEKRIPHEVVHRVPMPDGRIKVVVERCETAYDA EGKPIHSLGTVQDITEYYKQETILKKLLAGTSNTLTQEFFSALVRHIAEA LEVSYVIIAELIDERLHTFAFWGDEQLQKNIDVAICQTPCEYVIKDGFFY CSHSIQEQFPQNTHLAQMQAESYLGIVLTDKNSHPIGILCVLDVKPMDRE TAEMIQQILQIFAGRASAELERKRSDEALQQLKATLEAQVEERTQQLQES QRFIQQITDQSPSILYLYDLQEQRNIYINQEVSRILGYSPTEIQEMGNLI ISRLIHPQDLSRFNRYLEQLKQAQDHEILGVEYRFQDIKGQWRWFSGRDA VFSRDSQGRVKQVIGVAQDITERKQAEQTLYLQAQQEKLLREINQRIRQS LDLQTIFDTACQEILLLLQVDRVGIFRFDPESHYDDGEFIAEAMVAGLPS AIAIHVHDHCFGEKFSSLYAQGKFLAVDDINNSELMDCHREILSQFQIKA HLVLPLLCEEQLWGLLCVHQCYDTRHWKEAEIKLLQQITHQLTIAIQQAS LYEQIRQKLRQQQAIAAIVQQVRQSLNIEEILNTITQDVRALFDC DRVII FRLYSDGGSRIIEESVSTEFLPLKYCHWDDETWSQDILNLYWQGQPRIVP DVMNDIYTECLHEYSREGQIQSKIVAPILLDLKEKENHRWVASTNSHKLW GILVVH ACREKRVWQNSEAQLLQQIANQLAIAIQQASLFEQLQVEIEDKQ QKNAELDRATRLKDEFLANMSHELRTPLNAILGMTEGLQDEIFGQINERQ RKSLKIIEQAGNHLLELINDILDVSKIESGQLELHCTSTEIIPLCQSSLA FVKQQAVKKRIQLDFNISSNILMLTLDERRIRQVIINLLNNAVKFTPEGG KVGLEVVQIGENTVRFAVKDTGIGIAAENIPKLFQPFMQIDSALNRQYTG TGLGLALVKRLVDLHGGEVSVTSELGVGSCFSVDLPLMESCSTDNFFDFQ TPLTPEVEANSVNLKNAPLILLAEDNETNITTISNYLKAKKYKLILAKNG KEAISLAQSQQPDLILMDICLPGINGLEAIQQIRQLPDLKDIPIIAVTAL ALTGDRERCLEAGANEYLSKPLKLKELVALIQSLLE Spi9445_1327 (Bold and underlined region = SEQ ID NO: 19) (SEQ ID NO: 47) MQSHSFSSIDVTEAICPRPHVISPTATVLEAIALMSGLSAPEVAPPDPQD PHSTDLSPLAPLWKGGKEGGEEGGGISSCVLVVVERDSDETQGQRVVGIL TERDIVRLSAQQQDIRELSVGEAMTQPVLTLRPSELTDIFSLLQFLEQHH LRHVPIVDEQERLMGLISHETLRNLARPVDLLRLRSVQEVMTQTVLTASP DASLLEIAQLLAENRLSSVILTRPLAGGDLGEYPVGIVTERDVVQFQALG GNFGEILAEEVMSSPLFTLRPDADLWTAQQAMEKRRIRRVVVTGEGGELL GIVTQTSLLRSFNPLELYRLAEVLEQKVARLEAERVTLLERRTQELEQQV QERTQTIEAQAERVRLLLDIATSLRNSLDLGTILQTAVDEVRRVLECDRV MIYQLEEGLRGEIIAESMISGGRSVLHREANDPCVTPEWLESYRQGRVRV VRDIYEESLSLCHQEMLLSFEIRAKLMVPIVLEEHLWGLMIASYRDQPRD WQTWEVELLQALSLQLAIALQQAGQHQQLQNEIRERQQAEQDLAALNAQL EARVAQRTAELESREARYHALMEGASDAILLATPQGYIIEANAAAEELFG YSRSELTQLHYSQLCPPEELQPVTQVWQSLVNPQQRVLWDGFILHAEGHS IPIALSGTMIEVGDSIIFQGIFRDISARQQAEAALAKLSQRLSIALSSAA LGCWEWDIAQNCLTWDKRMYALYGVESRVPPDDPSTVTVAYEVWSKGVHP EDRQRTETLLQQALLGEAEYNTEFRVVHPDGSLHYIRAYGVVLRDAAGHP QSMIGVNLDVTDTHEAQRELQASETRFRQVFDSNVVGMMFTNFVGEITEA NDRFLAMLGYSRDDLHAGRLNWADLTPPEYQQQDVEAIYHLLTYNSIDPF EKVYLHRDGHPVAVLLGVAMVCPAEGTCVCVVVDISDRKQAEIALQESQL RLELALESSNTGLWDWNMQTGELWFNKQWKTMLGYGEDELENQLREWESR VHPDDLPQTYQEVEQHIKGQTDVYRNEHRLRGKDGSYHWVLAQGRIVERD GVGNPLRFIGTHTDISDRKNNELERQKLLQELSSFKFALDQSAIVVTTNL KGQILYINDRFESISGYSQPEILGKTPQILNSKYHPPGFFAHLWTTILNG QVWRNEICNRAKNGQIYWVDATIIPFLNPQGQPTQFLSIQFDITSRKQVE LDLASSNSLLSTITHAQAQFITAANRLTIFEGLLESLLELTHSEYGFIGE VLFQGDGTAHMEENFLKIRGVPYLQTHSITNIAWDAATEQFYQNNYEKGM EFTNLKTLFGAVILTGKPVIANQAPTDPRRGGIPKGHPPLEAFLGIPFFK GPELIGMVGIANRPGGYNEGIIARLGPFLTTCSNLIEGYRMDRHRQKAEA MIAQQLRQRTVLGQIVQQIRESLNLQEILAITTQRVREILQG DRVIVFRF CDLGRTCIFEEAVAEDLPSLKYMNWEDEQWSSEILQFYWQGQPRIVPDVM NDPLTPCLLDYSRQGQIQSKIVAPILQEIHNGERGNGEIDPWTDPESGNK LWGLLVIH ACHEKRIWQESEAELLQQIANQLAIAIRQSRLFEQLQEELTE RQQTQIQLTQRNEELIRATRLKDEFLANMSHELRTPLNAILGMTEGLQDG VFGSVNEGQRKALSTIERSGSHLLALINDILDLAKIESGQVELECAPTAI ASLCQSSITFVKQQALKKHLHLSVNLPVNLPDIVLDERRIRQVLINLLNN AVKFTPEGGRVTLEVTLPTPEQNSLPHLRFSVIDTGIGITPENLKKLFQP FIQIDSALNRQYQGTGLGLAVTKRIVELHGGQVGVSSEEGKGSCFMIDLP YQASVVFAPQTNSESHFDPHDLATQSPGKSSPLLLLAEDNEANISTISSY LMAKGYRIEVAKNGQEAIHQAVALSPDLILMDVQMPGMDGLEAMKRMREI PELATTPIIALTALAMDSDRDRCLQAGADEYLSKPVKLKQLTLTIQGLLK S Sta7437_1656 (Bold and underlined region = SEQ ID NO: 4) (SEQ ID NO: 48) MPLALSQIFHRLIANVPLRWVLTIPFVLPTIGAVAIVGYLSYRDGQEAVE DLGHQLVAETNERVKQELETYLQTPVLINRLNVDAVARGQLDLQNIVALE AVLFARLQQFERVSAVLFASPQGTFRLVDRLPDLYLVVADPPRPEQILIY SLNSDGSRKELVRTNEGLDVRRDNPWYRRAVRTGKPGWSPIAQYGSLNFL TLDASQPVYDRTTKSLLGVFAVHIRLDYLSEFLHHLDISRSGRVIIMDRN GALIATSTEEQPYKFLAGTGYQRQFEQINIDESQDNLTRSLGKYLRKRPE ILKSLERTRLLDFRYNGELQLVQIAPFQDQYGLNWQIVTVIPKSHFLKDI QENKRTTALLCLLTLGVALALGLVAADKLTASFARLSRVSRELAAGNLAR RLPTDSSIYELNGLAQTFNQMADQLQQSFDRIQIALEESEEKFATVFRTS PDPMAIASLAEGRILEVNDSHVDFFGYSRAETIGRTVLVLNLWSNLDERE KFRALLHQQGSVRNLEAQLRTKSGEVRTVLVSAEVQTLEGQDCTIIVLRD ISERKQAQAALQESETRFRQLAETVREGFFVYETKSDHYSYVNPAYAAIM GTPAQLFYQGMFHWLNNIHPDDCDHIEAGLLREHQGENFDEEYRFIRPNG EIRWLRSKAFPLRDETKTIVRIVGTVEDITERKQLEQSLRSQAEEERLIT TITQNIRQSLDLKKILATTVIEVQQTLNA ERVLIFRMNPDGSGQVIEEAV VPKYPVTDQMRWEDEHFPEDCYEYYRQGIPRIVPDVATDEWAKCLVEFMQ EVGVKSKVVAPIVQVYEKSSTNAKVWGLLIVH ACSHYRQWQESEVDFLQR IGNQLAIAINQANLYQQLQAELAERQQTEEAFRESEELFRRAFDDAPIGI ALVSPTGQFLKANTYYCNLLEYSEEELLTLTFQNITHPTDLEADFEVFRQ MMAGEIRSYHLEKRYITKQGIVIPVLLNAASIRDQDDRPLYCVGQIQDIR DRLKVERMKDEFISVVSHELRTPLTSIRGALGILGSGVFDNRPEKAKHML QIAINNSDRLVRLVDDILSLERLESGKVQLVMEQCQVAELMQQAIDSLQA LAERADLTLSVTPISATLWAAPDAIIQTLTNLLSNAIKFSSPGDTVWLKA EIGSGEWATANGQQFSDTQTPYILFTVKDRGRGIPEDKLEIIFEQFQQVD VSDSRQKGGTGLGLSICKRIVQQHGGRIWVESSLGEGSTFYFTLPIKEEN D MicvaDRAFT_3059 (Bold and underlined region = SEQ ID NO: 16) (SEQ ID NO: 49) MFMRTTALTPIELRTAIVREPLVVSPDTTVMDAIAQMSGVRSLCNTTRTA DGQLDDLHLEARSSCVLVVENEQLVGVLTERDVVRLSAQQRSLENLVLRE VMAHPVVTLRESAFTDLFFAINLLQQHHIRHLPILDDLDRLVGLVTHESL RQTSRPIDLLRLRMVAEVMTREVICAAPDSSLLAIAQLMAENRVSSVVIV HPGGISTEPLQIPVGILTERDIVQFQTLGLNLETCLAQAVMSTPIFAVRP DDSLWTVQEIVEQRSIRRLAVTGELGELLGIVTQTSLLQALNPLELYKLV QKWEEKVVRLEAEKVALLANRNVELEQQVEARTAALKAKADREQLLNTIA EQIRSSLNLSDILQTTVQEIHSLLGCDRVIIYQFRSDFSGTVIAEAITDT GRSVLHREAHDPCMSPEWLEPYRQGRIRIINDIYGEPMTQCHQEMLVGFD IRAKLMVPIVIEEQLRGLMIASYRASAHSWTTDEIELLRQVSLQVAIALG QAMIQQKLQNELVKRQRIEATLIESEQRYAALAAAAPVGIFRTDATGLCT YVNDRYFQISGLTPGATIGHGWQQGVHPDDRDWVMVEWKQFIQGNRSFEL EYRFQCPDGTVTWVYGQCVAELDANGHRSGYIGTITDISARKRTEVCLQE SEERYATLVAAAPVGIFRADAVGNCIYVNDRWCQISGLTPKTAVGEGWQQ GLHPDDRDCVIAEWEQSVQRNRPFQLEYRFQRPDGGVTSVYGQSVAERDA DGQVVGYVGTTTDITDRKQAEQKLQQLNQQLETKVAERTQELWQVNSLQR AILDCADYSIISSDPSGIIQTLNAAGERMLGYSAQEIIGQATPALIHDAN EVIDRAASLSAELGQNIPPGFEVFVAKARQGLVSEEEWSYIRKDGSRFPV SLSITALKDVHQQIIGFLGIAKDISDRKRAEAELQKLSERLALSLKSGAI ASWEWNLGQNTILGDERMYELFAVTKPSDACQVYDFWANRLHPDDRIPTE TLLHQAVLGQAEYDTEYRIVHPDGSLHFIKAYGVVVRDAQSNPQSMIGVN FDISDRKQAELQRQQLIQELSAFKQALDQSAIVVITDREGVISYVNDRFC VVSGYSRDRLIGQTHRLVNSGYHPPAFFQDLWRTINSSQIWRGEICNLAK NGSLYWVATTIVPFLDEQGRPFQYLAIGFDITDRKLAEATLQQENTFRQQ IVENMAEGLCVFHQVEEFPFVRFTVWNQQMQAITGYTLEEINRLGWYQTL YPNLEDREQAIANCRQMQPIAVEREIQRQDGQRRTISISTSVLSGDDGHL YALALIQDITHRQQTERENRLLKERLEFLLASSPAMIYSCKPYGDYELTF MSKNMSAILGYKPEEFLSESGFWANHLHPEDAPRVFADLSALFEYNTHQH EYRFLHHDGHYVWLRDELRVVRDEEGCPTEIIGYFADISDVKQTEETLKI QLAAIEAAIDGIAIMQGDTYLYLNQAHLELFGYEHPQELLGKTWQLLYSP EELERFEREVFPVLGRDRAWQGEAIGTRKDGSTFAEGLSLTLTENGLLIC VCRDISDRKQIEAELAESEAKFRRLVEGANDLIWSCEPDGILTYVSPQFK TMFGWDESAWIGKSFIYLVHPDDRSLVVTDYRENIKSGKKSSDYEFRHRH RDGNYVWVRSSATPVINAEGELISIQGILSDISDRKEAEIARESSEIRFR RVFESSVSGMIFADFQGNIIDANDRFLQMVGYTREELDAGLIHWDAMTPP EYFPADVLAMERVMQDGAIEPWEKEYYRKDGSRISVLIGVALLPDSDDQT ICVLVDISERKQAQKALQESQQFLQTVLDTIPLAVFWKNRESVFLGCNQQ FAQTLGLPSTTESIGKKDLDICQEEVEANEYCAMDRRLMETGEAILGIEE TLTLPNGKLIFIETHKAPLRDCSDNVIGLVGTFQDITDRKEAEQKLQQQA KQERLLGAITKRMRSSLNLDEILNSTVEEIHQLLQSDRTLVYRVFPEGTG AAIAESVSPNRLKLLDILFPEEVFPEDTYERYIQGRVYALNDSEDENESI VPCLVEFLADIEVRAKLVVPIIQNQTLWGLLIVHQCDRPRQWQDWEINLL KQIANQLAIAIQQSYLYEQVQSELAIRKQTENVIALQLQRQRTLGAIAQQ IRESLDINQILAAVTQQVKEILQG DRIIVFRLFGDGRSQIVEEAVSSEFP ALKDHHWEDERWSQEILNRYWQGKPRIVPNVMTDIWTDCLVEYASVGQVQ SKIVAPILQEVRSSESHRWIAPGQTKKLWGVLVVH ACREQRVWQESEAQL LQQIANQLAIAIQQASLFKQLQQELTERQQAQQQLTERNQQLGASNEELA RATRLKDEFLANMSHELRTPLNAILGMSEGLQEQVFGIVNEQQIKALQTI ERSSSHLLELINDILDVAKIESGQMELDCTPVSINHLCQSSLAFIKQQAL QKRIQLEIQMPLNLPDLLIDERRMRQVLINLLNNAVKFTPNGGRITLEVS RQQRPADPDSADSPPHFLVKETLRIAVIDTGIGIAPEHINKLFQPFIQID GALNRQYTGTGLGLALVKRIVELHGGQVGLTSTVGVGSCFTIDLPCTACA PSSVYLESQTEPRIEPSQPEEGGALP UYKDRAFT_01008 (WP_016871037; bold and underlined region = SEQ ID NO: 24) (SEQ ID NO: 50) MSKSPSHLLAEAEKAQVLGKFTEAEEYYEQAIDTAKANGSLQTEALAYEL AAKFYLERGRLRFAQNYIKEAHYAYTRLDARAKIKELETQYPQLRSELSA ADSHTSTDLEAVIRANQAIASEIELERSLSVLMKILIENAQAQTGYLILP CQTASTSTEKWAIAASGTIDIATNEQIIVLQSLAIADHLPASVIDYVIQT LESVVVDDATREGNFINDTYIQQHQTKSILCVPLLHQEELLGIVYLENNI TNGVFTKEQLKVIKLLSAQAAISLHNAKLYNQLRESEQQLRTREHRLNQI LEAMPIGVTAHNTNGEFIYSNLKAQQLLGITAPLEVTTEQLLQVFQVYQA GSDQLYPTDQLPIVRAFAGESVKIDDMELRQADKTVPLEVLTTPIFDETG AVIYAITAFTDITERKQAQKLLAKYNQTLEAQIAERTEKLQQQHEILQTL FDHMPVMLKLRDQTGQTVLINREYERVLGWSLREIRESDFLAECYPDLEQ RQRVEEHIQAATGKWQDFKTRCRDDRYVDTTWANIRLSNGWTVGIGKDIS DRKQLEAALQASKAKLKDILNSAQASIASFRVYPDGTWEPDYHSTGCETV FGYTPQEFTPAVWSSRVPAEDLAAIIEQRSTAIPKGEALTVEYRFYHKNG SLRWITETLTSRWDQVGGCWVVTMVAVDITAHKQAEQALQEAYRKLEEYS TNQEAVNQELQRTLEDLQVLEEERQEQNHQLLIEQQRYRDLFNFAPDGYL VTDAQGRILEANHAIATLLSVESGFLTGKLLVSFIPASARRAFRTQLNHL SSLPDKQTWELSLQPRQGEPFPVEITVAPVRDAQKLIALRWLIRDITERK QAETALRESEERFREIAENINQLFFVWSADSQQFLYISPGYEKIYGLTCE SLYQNSRSWLEVVHPDDRPSVLQSLDQQYQGKHAQREYRIIKSDGTIRWM FAEVFPIFDQTGNLLRYIGLTEDITERKRAEEALRQREQEFRALVENAPD VISRVDREYRFCYINPRVELETGIPPAQWIGKTELEMGFPQTIVNPWHAA LEHVFETKQEQIYEAEFPCPEGISYWLCRLVPELAEDGSVATVLSIARNI TDRKRAEEALRESEQFLRSIYEGIAAGVCIVDVLEDGSFRYVGINPAHER MSGLLSAEVAGKTPEQVFSPEDAQAVTARYRACIIARERITYEERLVFKG KETWWITNLSPLQNENGQIYRLIGSCFNITRRKKLEQSLQLQAEQERLLI TITQHIRQSLDLEQILRTTVVEVQRTLQT DRVLIFRLNQDGSGQIIEEAV VPEYPMTYQMRWVDECFPDDCYEYYRQGNPRILPDVAKDENGACLVEFMQ QIGVKSKVVAPIIQTLEDSSTRVWGLLIVH ACSHYRQWQASEAEFLQQIS NQLAIAIHQADLYYQLQIELAERKQMQLVLQERQAILRAIGDNLPKGFIF QIVHVPDQGVYFSYISAGIEDLIGLKPEAIIQDANVLRNLIHEEDKPVRQ KLGLKSLKTLCIFEMQMRFRSLRGNIIWLDVRSTPRRLRDGRTVWDGVGI DITDIKQAEDALRRSEAHLAMAQKVAQIGSWEFDLQSQQINWSETTFHHW GIEIDQGEPSFAELLVRVHPEDREILKQHIERAITQGIPYAFDLRIVLPD GSIRYLDSRGEPLVNAQGQVIKLIGTSLDITARKQAEGALRESEERFRKA FNAAPIGMALVSPQGQFLKVNHSLCEIAGYTEAEMLTLTLKDVIHTDDLE ASLEAMQQMLANDIRLYQVEKRSLHKQGDVIHILLNVSLVKDQHRQPLYF IVQIQDISDRYKVDRMKNEFISIVSHELRTPLTAIRGSLGILETGIFDHE PEQAKEMLQIAFNNSDRLVRLVNDILDLERLESGKTQLVMETCEIADLVQ QAIETVQAIAKEARVEISVMVANMQIWAAPDAIVQTLINLLSNAIKFSPV GGTVWISTEVLNQEMEKWKDREIGRKISPHHPTTPSPHFPNSHILFAVKD QGRGIPPEKLESIFGRFQQVDASDSRQKGGTGLGLSICKSIVDQHGGRIW VESLLGEGSTFYFILPLKRGEA UYEDRAFT_06529 (WP_016878855; bold and underlined region = SEQ ID NO: 25) (SEQ ID NO: 51) MPPDREKVGMGFDREEVSTNLQPQEALCARSESTKPKENILVVDDNPDDL DFLIQILSKHGYQVQLVPSGKLALIAVESTLPDLILLDIMMPEMDGFEVC SQLKASAQTKDIPIIFLSVLHKTFDKVKAFSLGAADYITKPFQPEEVLAR VENQLRIQRLTKQLVEEIKERNIAQEQLKNKEKHYRRLFEGSVDGIVLTD MQGRIIDCNASYQKMLGYSPEELKLLSFWDLTPIQWHCWEAEIVEQQIIE RGYSDTYEKEYIRKDGTIFPVELTVYCQKNDCGQPEIMWANVRDISDASR QAATRLRKQAQQALEQSIIKNRALLDAIPDMVFRCHVDGTYLEFKPAKDL KPFVPPSKFLGKKIQKILPDQVAQKILQAQQQAILLGETQILEYQLPIDG RLHDYEVRIVACGSHENILFVRDITERKLTEAALAKSEQKYRNLVETSQN IIMSCDRQGAITFVNQAVKQIYGYDPKEMIGHPFTDFLPPEIAAKDLEVF QQLLNGTPVNLYETTHRAKDGRLIHLLFNAIALFDEQGQVIGTTGTASDI TARKQTEEELQQAYKKLEEYNAELQATNQELQCMLEELQFFELERQQQYH QLIIEQKRYEDLFNFAPDGYLTTDATGIIQEANHAIAAYLSVDLKFLAGK PLANFISEGDRRAFRTQLNQLLSLQQKQTWELKLQPINGEPFAVEMTVAP VCGSSNQLISLRWLIRDITERKQAEAALRESEERFRQIAENIHQFFFVLS ADSGEYLYLSPAYEKIWGQSCESLYQNPKSWLEFVHPDDRQLVLHSLYQK NEGKRVQREYRIIRDDGTTRWIFAEVFPILAQSGELLRYVGLAEDITERK STEESLRESEHFLRSIYEGIEAAVFIVDVLEDGRFRYVGINPANERMSGL LSTEIAGRTPEQVLSPEDAQAVIDRYRTCVAARKPITYEESLVIQGKETW WITNLAPLQSEDGQIYRLIGTSFNITVRKQLEHFLRSQAQQERLLGTITQ HIRQSLNLEEILATTVIEVQQTLQA DRALIFQLNQDGSGQIIQEAVIPDY PVTNQMRWLDECFPDECYEYYCQGNARIVPDVAKDDWGACLVEFMQEVGV KSKVVAPIVQSFEGSSNKVWGLLIVH ACSHYRQWQASEVEFLQQLCNQLA IAIHQANLYHQLQIELVERKHTEKALQAAQESLTIAIEAAQMGTWHLDIT KDFASKRSLRHDQIFGYDTLQSEWGQKIARRHVVEEDREIFDAAFVRAME TGKLDFEVRIQWPDGSIHWMAARGRFYFDDNGKPVYGGGVNFDITDRKQT ELALRESEERFRRAFDDAAIGMAMVAIDGSFITVNRSLCEILGYSEAEFL ALTFQDITHADDLHKALDYRQRLLVGETRTYQTQKRYIHKLGHEVWILLS SSLVRERDGKPLYFINQYQDISDRQQISRMKNEFISIVSHELRTPLTAIR GSLGILETGVLKDEPQQAKELLQIALKNSNRLMRLVNDILDLERLESGKV RLIMQECEIGDLIKQATETVQAIADEANITLCATFPKIQIWAAPDAITQT LINLLGNAIKFSPVGSSVWLSAELFPDHVLFFVRDNGRGIPSDKLKTIFG RFQQVDASDSRQKGGTGLGLAICKTIIRQHGGKIWVESVLGEGSTFYFTL PFAQPDT Fis9431DRAFT_3998 (WP_026722600; bold and underlined region = SEQ ID NO: 26); (SEQ ID NO: 52) MITFLSHLIVEAEKAQVLGQVIATEEYYEQAIDAAKANNSLEQEASAYYE AAKYYLERGRPRVAQNYIKEAHYAYKCLDATAKVKDLETKYPQLLFELSS ANSNTCTRSTSFQFSSNASGEALELLEAVTRANVAISSEIELERLLRILM KILIENTDAHTGYLILPASTNLENGEEWEIAASGTIDTEASEDALGKPVL QISVQPLAIADHLPISVIDYIIHTLENVVVDDASCEGKFIHDSYIKEHQI KSILCVPLLNQGQLIGIVYLENNLTQGVFTKKELNILNLLFVQAAISISH AKIYKQLRESEKQLRAREKRINQILDAIPIGVTAHDPTGRFIYSNLKAQQ LLGIKTPPEIKIEQLSEAFQVYRAGTDEFYPIEQLPLIRAFAGESVKSDD MELRQVDKSIPLEVLTVPIFDGEGAVIYAIAAFKDITERKQAQKILADYN YTLEAQIVARTEKLQQQNEILQALFDHIPVMLKIRDQADQTLLINQEYEH TLGWTLEEMRDVDWLAKCYPDAEQRQQITEHIQAATGKWQDFRTRCHNGR YIDTSWANIRLSTGQIIGIGQDISDRKELEKALQASQAKLNDILNSAGAS IASFRVYPDRTWENEYHSLGCETVFGYSPEELTSELWLSRVPSQDLAAIT EQAFAAIAQEQAITVEYRFYHKNCSLRWIAHTLTSRWDQAEGCWIVTMVG VDISDRKQTEEELQQAYKQLEEYSADLEAINQELHLTLEHLQVLEEERRE QHHRLMHEQQRYQELFNFAPDGYLLTDARGTIQEANCAITALLSIELGYL IGKPLVSFIPASARRTFRTQLNHLSLLSDKQTWELSLRPRNGKPFPAEIT VAPVRDGNKLIALRWLIRDITARKQAEIALRESEERFREIAENINQIFFV WSANSEQFLYISPGYEKIYGMSCESLYQNPQSWLDLVHPDDRKSVWQSLN EQSQGKPARREYRIIKSDGTIGWMFAEVFPIFDQTGKILRYIGLTEDITE RKRAEEALLEREQFLRSIYDGTAAAIFIVDVLEDGSFRYVDINPAYEWMS GLLSSEIVGKTPEQIFPLEEAQVISARFHNCATTGTRIPYEERLLIRDKE TWWINVLTPIQREDGQIYRLIGSCFNITKRKKLEQSLRSQADQERLLITI TQHIRQSLDLEQILATTVIEVQQMLQVDRALIFRLNEDGSGQVIKEAVVP EYPVTEQMRWRDEPLPDYCYDFYRQGNPRIVPNVAIYDWASCLAEFLQQA SVKSKIVAPIVQTLEDSSTRVWGLLIVHACSDYRQWEASEAEFLQQISNQ LAIALHQANLYQQLQTELAERKQTEEALRQNQAHLAMAQKVSQIGSWEFD LNSQKIRWSQITFHHWGLEPAKGEPSFTELLAKVHPQDREVLQQNVEQAI AKGIPYTFDLRILWPDGSIRYLDSRAEPVFNAQGEVIHLIGTSLDITERK QAEERLRESEERFRKAFDAAPIGVALVSPQGQFLKVNRSLCEIVGYTEAE MLHLTMTEITHPDDLEADLEFIQKLLANEIRVYQVEKRYLHQRGDTIYIR LNVSLVKDRHRKPLYFIAQIQDISDRYEVDRMKNEFISIVSHELRTPLTA IRGSVGLLEEGVFDNEPEQAREMLQIACNHCDRLVRLLDEILDLERLESG KVQLVMETCEIANLIQLAIGTVQTTANQARVEISVVIVPNMQISAEADSI IRALTNLLSNAIKFSPAGSTVWLSAELLTPEEDAGIEGQGGKEGQIAPAS PVSPISPVSPISPMSPVSPVSPVSPMSPISPVSPISPVSPISPMSPMSPV SPMSPVSPQSPQILFKIRDQGRGIPPDKLESIFERFQQVDVSDRRQKQGT GLGLAICKNIVQQHGGHIWVESVLGEGSTFYFTLPITREEDC PCC9339DRAFT_00524 (WP_017309337; bold and underlined region = SEQ ID NO: 27) (SEQ ID NO: 53) MITFLSHLIVEADKARVLGQVIAAEEYYEQAIDGAKANASLEEEALAYEL AAKYYLERGRPRFAQNYMKESYYAYRRLDATAKVKELETKYPRLLFELSS ANSNTSTCFTSPKMSSISSEGALESLEAVIRANIAISSEIELERLLRVLI KILIENADAQTGYLILPSPTNLENGEEWKIAASGIIDTEASDNTLGKPVF KIGVQSLPIDDHLPTSIINYVIHTLENVVVDNASCEGKFIHDPYIQQHQT KSILCTPLLNQDKLIGIVYLENNLTNGVFTKTQLNILQLLFTQAAISIHN AKIFSQLRENEKQLSVREKRVNQILNVMPIAVTAHDTTGRYIYSNLKAQQ LVGMKAPLEIKTEQLSEVFQVYQAGTDQLYPINKLPVVRTFAGESVKIND MELRQDDKTIPLEVLTVPIFDETGAIIYAIAAFSDITERKQAQKLLADYN QTLETQIAERTEKLQQQNEILQALFDHIPVMLKLRDQTDQTLVINREYER VLGWTLDDLRDIDWLAKCYPDTEQRQQIREHIEAATGKWQDFRTRCQNGR YVDTTWANIRLSTGQIIGIGKDISDRKQLEKALQASQAKLNDILNSAGAS IASFRVYPDRSWDREYHSLGCENIFGYTPQELTPELWLSRVPSEDLTVIS EQAFAAIAQEQATTLEYRFYHKNSSLRWIADTLTSRWDQAGGCWIVTMVG VDITARKQAEMALQESEQFLRSIYEGTAAAIFIVDVLEDGRFRYVDINPA HEWMSGLFSSEMAGKTPEQIFPPEDAQVINARFVACTTIGQRITYEERLE IRGKETWWINVLTPIYTEDGQIYRLIGSCFNITRRKKLEHSLRSQADQEH LLGTITQHIRQSLDLEQILATTVVEVQRTLQA DRALIFRLNQDGSGQVIK EAVVPEYPMTSQMRCTDECFPDDCYEYYRQGNARILPDVAKDEWSDCLVE FMQQIGVKSKVVAPIIQTLEDSSTRVWGLLIVH ACSNYRHWRASEAEFLQ QISNQLAIALHQANLYNHLQTELAEHKQTEAALRQNQAHLAMAQKVSQIG SWEFDVNSQNISCSQTTFHQWGIEPVKGEPSFSELLERVHPDDREVLQQK VEQAITNRISYAFDLRIMRPDGSIRYLDSRAEPVLNAQGQVIQLIGTSLD ITERKQAEEYLRESEERFRKAFDAAPIGVALVSPQGQFLMVNHSLCEIVG YTEAEMLNLTMMEITHPDDLEADLELMQKLLANEIRVYQVEKRYLHNRGD TIHTLLNVSLVRDQHREPLYFIAQIQDISDRYEVDRIKNEFISIVSHELR TPLTAIRGAVGILETGVFDHEPEQAREMLQIAFNNSDRLVRLVNDILDLE RLESGKIQLVTETCETANLVKQAIETVQAMANEAGVKIFVMVPNMQISAA ADSIIQTLINLLSNAIKFSPAGGTVWLSAELVSPEEEGGMGGDGGGGGGG GGGGDGGDGGDGGDGGQIAPISAKILFKVRDQGRGIPPEKLESIFGRFQQ VDVSDRRQKRGTGLGLAICKNIVQQHGGCIWVESVLGEGSTFYFTLPITR EEA UYEDRAFT_00976 (WP_016873240; bold and underlined region = SEQ ID NO: 28) (SEQ ID NO: 54) MIDSANSSQRLQKYFAKIPLWLLLVVPFVLQLLATVGVIGYLCYEAWQRS THKVANQVMKEVGDRVQHYLSDYLETPQLINRLNANATDLNQIDINDPNS LESHFLQQIQAFNSVSRIHFSNPQGGYISAGNDERGLSVAFTENFVRGTL HVYGVDNQGKRTQQFVHQQNYDATKRPFYQAAAKASKPIWTPIYVYIPAS TGLGIAASYPLYDQLNRLQGVLSTDLTLANINQFLSNLKIGTQGKVLILE RSGLIVASSTSEKPFFISSNQRQTIRLKATESQEPLIRFTAQHLVSYFGD LTKIKTPEQLQFEVKGKRLFLQVNPFTDRFGLDWLIVTVLPESDLIADLD GNTQRMMLLSGFTLLLAIGTGILTACWIARPIRRLKKAAQAITKGQLNYP IATGGIGEVAQLAQGFQVMANQLDSSFRALKASEQKFATLLSNVPIGISV FDAKENPVLINKVGEEILGRGLVSDISFAQHSEVYQIYVAGTDQLYPTEQ LPATRGLRGETALIDDMEIEVNGRRIPLEVHTIPVFDDDSNVIYAINAFR DITQRRQAEKLWTDYEQELKCRVAEKTAALRQSEERFRLAVNHAPDVFVI YDRDRRFLYVNEKARELTGWTLDHFIGYRDDDLFPPEVTAPYLPILQKTI STKTLQIGECTIKLPEQKPSTFIVKYVPLLDEQGEIQQILGMTFDISDRK QIEEILRQSEARLTMAQRVAQVGSWEFDLNSQKMTWSEETFYHWGFDSTP EEPSYTELLKRVHPEDREILNYFFEQAIAQGIPYVLDLRIVRPDGSIRYL DYRGEPLFNAQGQVIKLIGTSVDISDRKWTEEALRQSEALNRAIVNALPD LIIRMHRDGTYLDVKPTTAFLTSASPLVVGLNVQAVLSPQVAQRRIAAIE NALQTGEIQVYEFPFVIQGQSLWQEVRVMPLDVDEVLVVIRDLTERKKAE EAVRLQAKREQLLRGITQRIRQSLDLEQILATTVNEVLQTLQS DRALIFR LHGNGTGQVIQEAVRPEYPVTEQMLFPDECFPQECYEYYCQGQPRIVSDV FAEDFSSCLVEFMQKIGVKSKIVAPIVQTTENSSTKVWGLLIVH ACSQHR QWQQSEADFLQQISNQLAIAIQQSQLYQQTRQQAQREQTLNRVVQAIRNS LDLDTIFATTVSEVGLLLQVMRVNIMQYLPERGIWVSAADYVQDPSLGNT VGFEIPDTSNPIATKIKQFEIVQIINDVASEEEIAQTYQGACLIVPLKVE QQIWGSLTLVKDPPSAWQQFEVDLTIAVADQLAIAIQQANFYNQLQIELT EHCQTEEALRRSEEQFRKAFDNAPIGMALVSLKGQFLKVNNSLCEILGYN GEELLALTFADITHPDDLEPDLESRRQILAGEIRVYQAEKRYLHSSGNTI HVLLKISLVRDQQRQPLYFIAQIQDISDRYKINRMKDEFVSIVSHELRTP LTAIRGSLGILETGVLDHDPEQIKELLQIALNNSDRLMRLVNDILDLERL ESGKVKLVMEACEVANLVKQATESVQAIADEANITLSVKFSNIQIWVAPD AIVQTLINLLSNAIKFSPAGSTVWLSAEEGIGDREQVTGDRGQGIGDKEK IFDNFSASSRSPCILFTVRDQGRGIPSDKLETIFERFQQVDVSDSRSKGG TGLGLAICKSIVKQHGGKIWVESRVGEGSTFYFTLPITRK 

What is claimed is:
 1. A protein fusion construct comprising a far-red cyanobacteriochrome (CBCR) domain linked to a heterologous domain, wherein the far-red CBCR domain comprises a CBCR polypeptide and a tetrapyrrole chromophore.
 2. The protein fusion construct of claim 1, wherein the CBCR polypeptide comprises a GAF domain having an acidic motif comprising: a conserved tryptophan residue; followed by two acidic amino acid residues, wherein at least one of the amino acid residues is an acidic amino acid residue; followed by a conserved glutamic acid residue; followed by a further amino acid residue; followed by an aromatic amino acid residue.
 3. The protein fusion construct of claim 2, wherein the acidic motif comprises an amino acid sequence set forth in SEQ ID NO: 1: (1) W-X⁹-X⁶-E-X¹-X⁵

wherein: W is a tryptophan residue; E is a glutamic acid residue; X¹ is an independently selected amino acid residue; X⁵ is independently selected from the group consisting of a phenylalanine residue, a tyrosine residue, and a tryptophan residue; X⁶ is independently selected from the group consisting of an aspartic acid residue and an asparagine residue; and X⁹ is independently selected from the group consisting of an aspartic acid residue and a glutamine residue.
 4. The protein fusion construct of claim 1, wherein the CBCR polypeptide comprises a GAF domain having an acidic motif comprising: a hydrophobic residue independently selected from the group consisting of an alanine residue, a cysteine residue, a valine residue, a threonine residue, a leucine residue, an isoleucine residue, a methionine residue, a phenylalanine residue, a tyrosine residue, and a tryptophan residue; followed by a further amino acid residue; followed by a conserved aspartic acid residue; followed by a conserved glutamic acid residue; followed by a further amino acid residue; followed by a hydrophobic residue independently selected from the group consisting of an alanine residue, a cysteine residue, a valine residue, a threonine residue, a leucine residue, an isoleucine residue, a methionine residue, a phenylalanine residue, a tyrosine residue, and a tryptophan residue; followed by a proline residue.
 5. The protein fusion construct of claim 4, wherein the acidic motif comprises an amino acid sequence set forth in SEQ ID NO: 2: (2) X²-X¹-D-E-X¹-X²-P

wherein: each X² is independently selected from the group consisting of an alanine residue, a cysteine residue, a valine residue, a threonine residue, a leucine residue, an isoleucine residue, a methionine residue, a phenylalanine residue, a tyrosine residue, and a tryptophan residue; X¹ is an independently selected amino acid residue; D is an aspartic acid residue; E is a glutamic acid residue; and P is a proline residue.
 6. The protein fusion construct of claim 1, wherein the CBCR polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 3: (3) X⁹-R-X¹-X³-X⁴-F-X¹-X³-(X¹)₂-X⁶-G-(X¹)₃- X⁴-X²-E-E-X¹-V-(X¹)₃-X²-(X¹)₂-X²-(X¹)₄- W-X⁸-X⁶-E-X¹-X⁵-X¹-X⁷-X⁹-(X²)₂-X⁸-X²- Y-X¹-Q-G-X¹-P-R-I-V-X¹-X⁶-V-X²-X¹⁰-X¹-D-X¹-X⁵- X²-X¹-C-L-X¹-E-X⁵-(X¹)₅-X⁴-X¹-S-K-X⁴-V-A-P-I-X²;

wherein each A is an alanine residue, each C is a cysteine residue, each D is an aspartic acid residue, each E is a glutamic acid residue, each F is a phenylalanine residue, each G is a glycine residue, each I is an isoleucine residue, each K is a lysine residue, each L is a leucine residue, each P is a proline residue, each Q is a glutamine residue, each R is an arginine residue, each S is a serine residue, each V is a valine residue, each W is a tryptophan residue, and each Y is a tyrosine residue; and wherein: each X¹ is an independently selected amino acid residue; each X² is independently selected from the group consisting of an alanine residue, a cysteine residue, a valine residue, a threonine residue, a leucine residue, an isoleucine residue, a methionine residue, a phenylalanine residue, a tyrosine residue, and a tryptophan residue; each X³ is independently selected from the group consisting of a valine residue, a leucine residue, an isoleucine residue, and a methionine residue; each X⁴ is independently selected from the group consisting of a valine residue, an isoleucine residue, and a threonine residue; each X⁵ is independently selected from the group consisting of a phenylalanine residue, a tyrosine residue, and a tryptophan residue; each X⁶ is independently selected from the group consisting of an aspartic acid residue and an asparagine residue; each X⁷ is independently selected from the group consisting of a glutamic acid residue and a glutamine residue; each X⁸ is independently selected from the group consisting of an aspartic acid residue, an asparagine residue, a glutamic acid residue, and a glutamine residue; each X⁹ is independently selected from the group consisting of an aspartic acid residue and a glutamate residue; and X¹⁰ is absent or X¹⁰ is one or more independently selected amino acid residues.
 7. The protein fusion construct of claim 6, wherein X¹⁰ is absent.
 8. The protein fusion construct of claim 1, wherein the CBCR polypeptide comprises an amino acid sequence set forth in: SEQ ID NO: 4 (Sta7437_1656), SEQ ID NO: 5 (Cy7822_4053g2); SEQ ID NO: 6 (Anacy_2551g3); SEQ ID NO: 7 (Anacy_4718g3); SEQ ID NO: 8 (N7104D_1016g3); SEQ ID NO: 9 (L6406D_1154g2); SEQ ID NO: 10 (c56D2_02270g2); SEQ ID NO: 11 (c407D_01196g2); SEQ ID NO: 12 (fdiDRAFT29700); SEQ ID NO: 13 (WP009627289g3); SEQ ID NO: 14 (Os7112_5903g3); SEQ ID NO: 15 (C6303_3693g3); SEQ ID NO: 16 (WP006632756g3); SEQ ID NO: 17 (Cy7425_1390g3); SEQ ID NO: 18 (WP017296986g2); SEQ ID NO: 19 (WP_033374293); SEQ ID NO: 20 (WP028089844g3); SEQ ID NO: 21 (WP008316973g2); SEQ ID NO: 22 (Ga0039499_10213); SEQ ID NO: 23 (310F_3509); SEQ ID NO: 24 (WP_016871037); SEQ ID NO: 25 (WP_016878855); SEQ ID NO: 26 (WP_026722600); SEQ ID NO: 27 (WP_017309337); or SEQ ID NO: 28 (WP_016873240).
 9. The protein fusion construct of claim 1, wherein the tetrapyrrole chromophore is a bilin.
 10. The protein fusion construct of claim 1, wherein the C-terminus and/or N-terminus of the CBCR polypeptide is linked to the heterologous domain.
 11. The protein fusion construct of claim 1, wherein the heterologous domain comprises a heterologous oligopeptide or a heterologous polypeptide.
 12. The protein fusion construct of claim 1, wherein the heterologous domain comprises a heterologous polypeptide selected from the group consisting of a signaling polypeptide, a structural polypeptide, a transport polypeptide, a targeting peptide, a hormone polypeptide, and a regulatory peptide.
 13. The protein fusion construct of claim 1, wherein the heterologous domain comprises a streptavidin polypeptide.
 14. A method for detecting a cellular component, the method comprising: providing a protein fusion construct in a sample, the fusion construct comprising a far-red CBCR domain and a heterologous domain specifically detecting a cellular component; exposing the protein fusion construct to far-red light or near-IR light, wherein the exposing causes fluorescence of the far-red CBCR domain; and detecting the fluorescence of the far-red CBCR domain, thereby detecting the cellular component.
 15. The method of claim 14, wherein the sample is a cell or tissue and wherein providing the protein fusion construct comprises expressing the protein fusion construct in the cell or tissue.
 16. A method for imaging a biological structure in a subject, the method comprising: providing a protein fusion construct in or near the biological structure, the fusion construct comprising a far-red CBCR domain and a heterologous domain; exposing the protein fusion construct to far-red light or near-IR light, wherein the exposing causes absorbance by, or release of an acoustic signal or fluorescence from, the far-red CBCR domain; detecting the absorbance, acoustic signal, or fluorescence of the far-red CBCR domain; and constructing an image of the biological structure; thereby imaging the biological structure.
 17. The method of claim 16, wherein the biological structure is selected from the group consisting of a tissue, an organ, or a tumor.
 18. The method of claim 14, wherein the far-red cyanobacteriochrome domain comprises a CBCR polypeptide and a tetrapyrrole chromophore.
 19. The method of claim 16, wherein the far-red cyanobacteriochrome domain comprises a CBCR polypeptide and a tetrapyrrole chromophore.
 20. The method of claim 18, wherein the CBCR polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOS: 1-28.
 21. The method of claim 19, wherein the CBCR polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOS: 1-28.
 22. The method of claim 18, wherein the tetrapyrrole chromophore is selected from the group consisting of phycocyanobilin (PCB), phytochromobilin (PΦB), phycoerythrobilin (PEB), and biliverdin (BV).
 23. The method of claim 19, wherein the tetrapyrrole chromophore is selected from the group consisting of phycocyanobilin (PCB), phytochromobilin (PΦB), phycoerythrobilin (PEB), and biliverdin (BV). 